Publications

154

Publications

Publications

155

List of publications

RESEARCH PUBLICATIONS AS PART OF THESIS.

1. L. Sagaya Selva kumar, K. Vasanth Ragavan, K.S. Abhijith and M.S.

Thakur. Gold Nanoparticle based immunodipstick biosensor for vitamin B12.

(Accepted in Analytical Methods. 2013, (IF.1.50)

2. L. Sagaya Selva kumar, M.S. Thakur.Nano RNA aptamer wire for analysis

of vitamin B12. Analytical. Biochemistry. 2012, 427, 151-157. (IF.3.236).

3. L.Sagaya Selva kumar, M.S. Thakur. Dipstick based

Immunochemiluminescence biosensor for the sensitive detection of vitamin

B12. A novel approach. Analytica. Chimica. Acta. 2012, 722:107-113. (IF.

4.310).

4. L. Sagaya Selva kumar, M.S. Thakur. Competitive immunoassay for

analysis of vitamin B12. Analytical. Biochemistry. 2011, 418:238-246.

(IF.3.236).

Book Chapter

5. L. Sagaya SelvaKumar and M. S. Thakur, 2012. Assay by biosensor and

chemiluminescence for vitamin B12. In: Series of Food and nutritional

components in focus. Published by the Royal Society of Chemistry (RSC) .

Nature –India Highlight

6. L Sagaya Selva kumar, M.S. Thakur 2012. VITAMIN SENSOR. Research Highlighted in NATURE INDIA. Doi:10.1038/nindia.2012.56;published online 20 April 2012.

OTHER PUBLICATIONS DURING MY RESEARCH WORK AT CFTRI.

7. U.S. Akshath, L. Sagaya Selva kumar, M.S. Thakur. Formaldehyde

detection using enhanced chemiluminescence. Analytical. Methods.

2012, 4:699-704. (IF.1.8).

8. A. Kumudha. L. Sagaya Selva Kumar. M.S. Thakur, G.A. Ravishankar, R.

Sarada. Purification, identification, and characterization of methylcobalamin

from Spirulina platensis. Journal of Agricultural Food Chemistry. 2010,

58:9925-9930 (IF.2.816).

9. L. Sagaya Selva kumar, R.S. Chouhan, M.S. Thakur. Enhancement of

chemiluminescence for vitamin B12 analysis. Analytical. Biochemistry. 2009,

388:312-316. (IF.3.236).

Publications

156

10. Neeraj Katiyar, L. Sagaya Selva kumar, Sanjuktha patra and M.S. Thakur.

Goldnanoparticle based colorimetric aptasensor for Theophylline.(Accepted

in Analytical Methods), 2012, (IF. 1.5)

11. K. Vasanth Ragavan, L. Sagaya Selva kumar and M.S. Thakur. Rapid

colorimetric aptasensor for onsite monitoring of Bisphenol-A in water

samples..(Communicated to Biosensor and Bioelectronics). 2012, (IF.5.6)

12. R. S. Chouhan, L. Sagaya Selva kumar and M. S. Thakur. Stabilization of

horseradish peroxidase conjugate for Immuno-chemiluminescence

analysis. 2010. (Communicated to J. Biotech. 2010. (IF.1.8).

Review

13. L. Sagaya Selva Kumar, R. S. Chouhan, M.S. Thakur. Trends in

analysis of vitamin B12. Analytical biochemistry. 398(2):139-49, 2010

(IF: 3.236)

Conference/Symposium Attended

1. K.V. Ragavan, L. Sagaya Selvakumar, M.S. Thakur. Conference: XXII.

Association of Food Scientists & Technologists. XXII-ICFoST-2012. (Dec 6-7). (Best Poster Award).

2. Neeraj Katiyar, L. Sagaya Selvakumar, M.S. Thakur. Conference: 3rd International Conference and Exhibition on Analytical & Bioanalytical Techniques. XXII-ICFoST-2012. (Nov 22-24). (Best Poster Award). 3. L. Sagaya Selvakumar, M.S. Thakur. Conference: Vitamin B12

Symposium, Nancy, France (20-22nd September, 2012).

4. L. Sagaya Selvakumar, M.S. Thakur. Conference: Indo Swiss

Collaboration in Biotechnology.ISCB-2011, Delhi, India (10-11th March,

2011).

5. L. Sagaya Selvakumar, M.S. Thakur. Conference: 16th International Symposium on Bioluminescence and Chemiluminescence.ISBC-2010. (April 19-23rd, 2010 ).

6. L. Sagaya Selvakumar, R.S. Chouhan, M.S. Thakur. Conference: 6th

international Food Convention. IFCON-2008. (Dec 15-19).

7. L. Sagaya Selvakumar, R.S. Chouhan, M.S. Thakur. Conference: National

symposium/workshop on new trends of biosensor technology, NSNTBT-

2009. (Jan 15-19).

Competitive immunoassay for analysis of vitamin B12

L. Sagaya Selva Kumar, M.S. Thakur ⇑Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (A Constituent Laboratory of the Council of Scientificand Industrial Research [CSIR]), Mysore 570 020, India

a r t i c l e i n f o

Article history:Received 14 May 2011Received in revised form 10 July 2011Accepted 11 July 2011Available online 21 July 2011

Keywords:Vitamin B12

IgY antibodyELISAHPLCPharmaceutical and food analysis

a b s t r a c t

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed forderivatized vitamin B12 by generating chicken egg yolk immunoglobulins (IgY) against derivatized vita-min B12 and purified using affinity chromatography. Checkerboard assay was performed with vitamin B12

antibody and vitamin B12–alkaline phosphatase conjugate followed by its conjugate characterizationusing ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit ofdetection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (Ka) ofthe vitamin B12 antibody was found to be 4.23 � 108 L/mol. Cross-reactivity with other water-solublevitamins was found to be less than 0.01% except for analogs of vitamin B12 that showed 12% to 35%.The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2%and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity,specificity, precision, and recovery of vitamin B12 with spiked multivitamin injections, tablets, capsules,and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000to 10,000 ng/ml. After extraction of vitamin B12 using Amberlite XAD, the developed ELISA method cor-related well with the established HPLC method with a correlation coefficient of 0.90.

� 2011 Elsevier Inc. All rights reserved.

Vitamin B12 is an organic complex that is included in a family ofcompounds called cobalamins that contains cobalt and is similar tothe structure of pigments such as chlorophyll and hemoglobin.Among them, cyanocobalamin, hydroxocobalamin, adenosylcobal-amin, and methylcobalamin are the major forms. Cyanocobalamin(vitamin B12),1 the most stable active cobalamin, is the form used forfortification. Vitamin B12 is an essential nutrient for whole cell devel-opment and human growth. It acts as a coenzyme for normal DNAsynthesis and plays an integral role in the development of the myelinsheath [1]. Deficiency of vitamin B12 leads to nerve degeneration,pernicious anemia, cardiovascular disease, weakness, and/or fatigue[2]. The chief source of vitamin B12 is liver, milk, meat, eggs, fish, oys-ters, and clams [3]. It is reported that a vegetarian diet lacks vitaminB12, and so vegetarians are commonly prone to the deficiency of vita-min B12 [4]. Excessive consumption of vitamin B12 may cause asthma

and folic acid deficiency; therefore, typically only a low level of vita-min B12 (e.g., ng/g) is added to products, making direct analysis dif-ficult [5]. The most common requirement for the analysis of vitaminB12 is in the quality control of pharmaceuticals (tablets or injections),blood plasma serum, milk products for infants, and fermentationproducts and involves complicated sample preparation [6]. The dailyrequirement of vitamin B12 is very low when compared with that ofother vitamins [7], and deficiencies are reported to be at the nano-gram level [8]. Extracting vitamin B12 from a larger amount of solidsamples is not so simple and effective; therefore, adsorption on char-coal or anion-exchange chromatography and solid-phase extractionsare the techniques usually used for concentrating the analyte fromliquid samples by eliminating most of the interfering compounds[9]. At the onset of these challenges, it is very important to diagnosevitamin B12 at the sensitive level.

Conventional methods for the analysis include microbiologicalassay, which uses Lactobacillus leichmannii as a test organism thatis very laborious [10]; the radioisotope method, which has beenapplied for the determination of vitamin B12 in food samples usingintrinsic factor as a recognition molecule, although the cost of theassay is quite high [11,12]; and high-performance liquid chroma-tography (HPLC), which has been employed by a number of work-ers to assay vitamin B12 in multivitamin and mineral tablets. Thesematrices are less complex than foodstuffs; therefore, extractionand resolution of the vitamin are simple in comparison [13–15].Other specific detection methods based on the dosage of cobalt,

0003-2697/$ - see front matter � 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2011.07.011

⇑ Corresponding author. Fax: +91 821 2517233.E-mail addresses: msthakur@cftri.res.in, msthakur@yahoo.com (M.S. Thakur).

1 Abbreviations used: vitamin B12, cyanocobalamin; HPLC, high-performance liquidchromatography; LOD, limit of detection; IgG, immunoglobulin G; IgY, egg yolkimmunoglobulin; ELISA, enzyme-linked immunosorbent assay; KLH, keyhole limpethemocyanin; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; SDS, sodium dodecyl sulfate; FCA, Freund’s completeadjuvant; FICA, Freund’s incomplete adjuvant; ALP, alkaline phosphatase; MWCO,molecular weight cutoff; UV, ultraviolet; OD, optical density; PBS, phosphate-bufferedsaline; PAGE, polyacrylamide gel electrophoresis; PBST, PBS with Tween; LDD, leastdetectable dose; MS/MS, tandem mass spectrometry.

Analytical Biochemistry 418 (2011) 238–246

Contents lists available at ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

either by atomic absorption spectrometry [16] or by inductivelycoupled argon plasma emission spectrometric detection [17], havebeen proposed. However, the limit of detection (LOD) of thesemethods (150 mg/g) allows only the quantification of highamounts of vitamin B12 in food samples. Similarly, methods suchas spectrophotometry [18,19], fluorimetric assay [13], capillaryelectrophoresis [20], and chemiluminescence [21] are found to beless dependable in analyzing a crude sample considering the time,cost, and sensitivity constraints. Such a situation demands morespecific methods such as biospecific assays that make use of bio-recognition molecules such as antibodies for the detection of vita-min B12 [22]. Development of an immunological-based method is,therefore, a promising solution; if care is taken, it is possible to ex-ploit the principle of antigen–antibody interactions. The problemof raising immunoglobulin G (IgG) antibody against the analytein question, which is a rather costly procedure, is resolved withthe development of efficient protocols for raising and extractingimmunoglobulins from hens’ egg yolk, namely IgY [23].

IgYs from immunized hens have considerable advantages forthe production of polyclonal antibodies because of the phyloge-netic distance between birds and mammals and noninvasive col-lection. In addition, there are several advantages of IgY antibodytechnology over conventional antibody production that uses rab-bits and other mammals. Housing for chickens is inexpensive,egg collection and storage are easier, and isolation of IgY antibodyis simple and fast. It has been suggested that the immunization ofhens represents an excellent alternative for the generation of poly-clonal antibodies [24–26]. The binding capacity and specificity ofdeveloped antibodies were checked using enzyme-linked immuno-sorbent assay (ELISA), which is recognized as a valuable tool in res-idue analysis and complements conventional analytical methods.ELISA provides rapid sample testing and accurate results, and itis more cost-effective than conventional chromatographic analysis[27]. ELISAs have been used successfully for the quantitative anal-ysis of numerous water-soluble vitamins in various matrices withlittle or no matrix interference [28–30]. Currently, there is no cost-effective immunochemical analytical method for the detection andquantitation of vitamin B12. In the current study, hens were used asimmunization hosts to raise IgY antibody against a derived form ofvitamin B12 by immunizing with vitamin B12 conjugated to a car-rier protein, keyhole limpet hemocyanin (KLH), because vitaminB12 itself is not immunogenic. Vitamin B12 was detected at a sensi-tive level of 10 ng/ml with an LOD 15-fold higher than with HPLCmethods and was successfully applied in pharmaceutical and se-lected food samples.

Materials and methods

Reagents and instrumentation

Vitamin B12, hydroxocobalamin, adenosylcobalamin, methylco-balamin, KLH, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimeth-ylaminopropyl) carbodiimide hydrochloride (EDC), sodiumcyanoborohydride, sodium borohydride (NaBH4), sodium dodecylsulfate (SDS), acrylamide, bisacrylamide, glycine, ammonium per-sulfate, tetramethylethylenediamine (TEMED), b-mercaptoethanol,bromophenol blue, Coomassie Brilliant Blue R-250, Freund’s com-plete adjuvant (FCA), and Freund’s incomplete adjuvant (FICA)were procured from Sigma (USA). Sepharose CL-4B was obtainedfrom Amersham Pharmacia Biotech (Sweden). Amberlite 53 XAD-2 was obtained from Supelco (Sigma–Aldrich, Bangalore, India).Alkaline phosphatase (ALP) and p-nitrophenyl phosphate wereprocured from Himedia Laboratories (Mumbai, India). Methanolwas of HPLC grade from RFCL (Bangalore, India), and all other re-agents used were of analytical grade. Vitamin B12 IgY antibody

used in this investigation was successfully raised in poultry inour laboratory. Amicon bioseparation centrifugal filter devices,with molecular weight cutoffs (MWCOs) of 30 and 100 kDa, wereprocured from Millipore (Bedford, MA, USA). An electrophoresisunit minislab was procured from Balaji Scientific (Chennai, India).The ELISA microtiter plate was a product of Tarsons (India), andELISA-based analysis was carried out using a VersaMax tunablemicroplate reader from Molecular Devices (USA). An ultraviolet/visible (UV/Vis) spectrophotometer (UV-160A) and a reverse-phaseHPLC system (SCL-10-AVP) were procured from Shimadzu (Kyoto,Japan). An HPLC column (Bondapak C18, 300 � 4.6 mm, 10 lm par-ticle size, 125 Å) was procured from Waters (Milford, MA, USA).Triple-distilled water was used for the preparation of the solventsystem for HPLC analysis.

Preparation of hapten–protein conjugates containing vitamin B12

epitopes

Vitamin B12 was conjugated to a carrier protein, KLH, and ALPby the mild acid hydrolysis method as shown in Fig. 1. VitaminB12 was subjected to mild acid hydrolysis, and the mono-, di-,and tricarboxylic derivatives of vitamin B12 were produced by dis-solving 10 mg of crystalline vitamin B12 in 10 ml of 1 M HCl atroom temperature, which was then covered and placed in the darkat room temperature for 4 h [31]. A total of 10 mg of carboxylderivative of vitamin B12 and 100 lg of NHS were dissolved in2 ml of water. During continuous stirring, the pH was adjusted to5.5 with 0.2 M NaOH and then maintained at this level. In 1 mlof water, 100 lg of EDC was added. After 3 min, 10 mg of KLHand ALP in 1 ml of water was added. The mixture was stirred over-night at room temperature in the dark. The conjugate was furtherpurified using 30-kDa Amicon Ultra filters, and conjugates werestored at 4 �C until further use. Conjugate confirmation was doneusing HPLC followed by its hapten density using UV spectroscopy.

UV spectroscopic characterization

Vitamin B12–KLH conjugate prepared for immunization wascharacterized with UV absorption spectroscopy. The concentrationof the KLH and immunogen was kept at 0.5 mg/ml based on the re-sults of a Bradford assay. The ratio was determined by taking theabsorption of the KLH and vitamin B12–KLH conjugate at 361 nm,which was the absorption maximum of the vitamin B12–KLH con-jugate. The absorption of KLH at 361 nm was deducted from that ofthe conjugate to find the absorption due to the vitamin B12 resi-dues. The hapten density, or number of moles of vitamin B12 permole of KLH, was calculated based on the following equation:

hapten density ðXÞ ¼ concentration of KLH in immunogen ðAÞ=molecular weight of KLH;

(where A = [(optical density (OD) of immunogen � OD of KLH at361 nm)/molar extinction coefficient of vitamin B12] �molecularweight of vitamin B12 [32].

HPLC confirmation of conjugate

Standard control KLH and conjugate were subjected to HPLC forits confirmation of conjugation at a wavelength of 200 nm.

Generation of vitamin B12 IgY antibody

Vitamin B12 IgY antibody was raised by immunizing a whiteLeghorn hen with vitamin B12–KLH conjugate subcutaneously.The initial dose of the hapten (1 mg/kg body weight) was adminis-tered using FCA. After a period of 21 days, booster doses were given

Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246 239

with FICA at a concentration of 0.5 mg/kg body weight at timeintervals of 2, 8, and 13 weeks. The eggs were collected and pro-cessed by the water dilution precipitation method to isolate vita-min B12 IgY antibody [23]. The titer development was monitoredafter each booster immunization. The concentration of IgY anti-body can be calculated from the absorbance value measured at280 nm: concentration = OD at 280 nm � dilution factor/extinctioncoefficient � path length [33].

Purification of vitamin B12 IgY antibody

Activation of Sepharose CL-4BActivation of Sepharose with sodium metaperiodate was carried

out for purification of antibodies [34]. With distilled water, 10 mlof Sepharose CL-4B gel was thoroughly washed, followed by treat-ment with 10 ml of 0.2 M sodium metaperiodate, and incubated at37 �C for 90 min with intermittent shaking. The gel was washedwith an equal quantity of glycerol (0.2 M) to stop the reactionand further washed with phosphate-buffered saline (PBS, 50 mM,pH 7.4). The activated gel was stored in 0.02% sodium azide solu-tion to prevent bacterial contamination at 4 �C until further use.

Hapten affinity chromatography for purification of vitamin B12 IgYantibody

Sepharose–KLH affinity column was prepared for purification ofvitamin B12 antibody by activation of Sepharose CL-4B gel beads bythe periodate activation method using 0.2 M sodium periodate.KLH was added to the activated Sepharose gel beads for immobili-zation. A concentration of approximately 10 mg/ml KLH was addedto the Sepharose gel volume [35]. Sodium cyanoborohydride(0.2 M) was used as a Schiff’s base reductant and incubated for16 h at 4 �C. Later, the immobilized gel was washed with PBS(50 mM, pH 7.5). The percentage binding of KLH with matrix wascalculated by comparing ODs before and after incubation of KLHwith matrix. Vitamin B12 IgY antibody was then purified using aKLH affinity column. A vitamin B12 IgY antibody concentration of5 mg/ml was initially loaded to the KLH affinity column, and elutedfractions were scanned at 280 nm after each cycle.

SDS–PAGE and pepsin digestion of IgYSDS–PAGE (polyacrylamide gel electrophoresis) was done for

isolated IgY under reducing conditions on 12% homogeneous gelusing an electrophoresis apparatus (minislab) according to themethod developed by Laemmli [36]. Gel was loaded with 50 lgof samples and run under denaturing conditions for 90 min at120 V. Coomassie blue staining was done using a standard devel-oping protocol supplied by the manufacturer. The gel was washed

three times in Milli-Q water, followed by destaining for 1 to 2 hwith destain reagent and then by washing in Milli-Q water. Diges-tion of IgY (2 mg/ml) with pepsin was performed in 50 mM sodiumacetate buffer at pHs between 4.2 and 5.5. Pepsin in the same buf-fer was added to give an enzyme/protein ratio of 1:100. Digestionat 37 �C was done and monitored by SDS–PAGE [37]. Digestion wasstopped by adjusting the pH to around 8.0 and further purified byan Amicon Ultra filter with an MWCO of 30 kDa.

Competitive direct ELISA for vitamin B12 determination

Checkerboard titrationTwo-dimensional titrations, in which various dilutions of the

purified antibody were titrated against various concentrationsof vitamin B12–ALP conjugates, were used to obtain an estimateof their appropriate concentrations for competitive assays. Amicrotiter plate was coated with 100 ll/well of the purified anti-body, serially diluted (1:10, 1:100, 1:1000, and 1:10,000) with50 mM carbonate and bicarbonate buffer (pH 9.6), and incubatedovernight at 4 �C in the dark. After the coating solution was re-moved, blocking solution (200 ll/well of 1% gelatin in PBS con-taining 0.1% NaN3) was added and the wells were washed withPBST (PBS with Tween) after 2 h of incubation at 37 �C in thedark. Next, 100 ll/well of the serially diluted standards(100,000, 10,000, 1000, 100, 10, and 1 ng/ml�1 vitamin B12 indouble-distilled water) or samples, along with 100 ll/well of seri-ally diluted vitamin B12–ALP conjugates (1:100, 1:10, 1:1, 1:0.1,and 1:0.01 diluted in PBST), were incubated for 1 h at 37 �C inthe dark. The wells were then washed three times using 200 llof PBST in the dark, and color development was done using p-nitrophenyl phosphate (1 mg/ml, 100 ll/well) in 1% diethanola-mine buffer (pH 9.8) at 37 �C for 30 min. The reaction wasstopped by adding 3 M NaOH (40 ll/well), and the absorbancewas read at 405 nm in an ELISA microplate reader. All analyseswere done in duplicates.

Calculation of affinity constant of purified vitamin B12 IgY antibodyThe affinity constants (Ka) of the purified vitamin B12 IgY anti-

body were estimated by Scatchard plot [38]. This is a simple andreliable method that is based on the law of mass action for calcu-lating the affinity constant of an antibody directed against the hap-tenic vitamin B12 group using the data obtained from competitiveELISA. It involves determination of the bound/free ratio at differentconcentrations using the following formula. The bound/free ratiocan be obtained from the displacement curve:

ci ¼ bound i=free i ¼ ½ðBi � NSB=BoÞ=ð100� boundÞ� � 100;

Fig.1. Reaction sequence for preparation of KLH–vitamin B12 conjugate.

240 Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246

where NSB = nonspecific binding, Bi = response value of differentstandards, and Bo = total response value or count.

The molar concentration of bound fraction [BnM] of enzymeconjugate is calculated as follows:

xi ¼ BnM ¼ bound i� ½molar concentration of antigen�=100:

Using linear regression, the new value of y (yc) is calculated againsteach x. The curve was plotted with yc on the y axis and BnM on the xaxis.

The equilibrium constant or affinity constant (Ka) is calculatedfrom the graph as follows:

Ka ¼ y-intercept=x-intercept:

Extraction of vitamin B12 from pharmaceutical and food samplesVitamin B12 injection ampoules having a concentration of

0.334 mg/ml were diluted to 10 ml with water, making a stocksolution of 100 lg/ml, and at least 6 of each capsule and tablet con-taining 15 lg of vitamin B12 were taken, equivalent to 90 lg ofvitamin B12, and diluted to 9 ml, giving a stock of 10 lg/ml, whichwas suspended in triple-distilled water for derivatization and di-luted for ELISA. But for food samples (multivitamin chocolate), atotal of 200 g (0.5 lg/100 g) was taken and suspended in distilledwater and centrifuged at 10,000g for 10 min. For further purifica-tion, the sample was loaded onto 30 g of Amberlite XAD-2, pre-pared as a methanolic suspension of the resin packed to a bedheight of 15 to 16 cm and a diameter of 2.5 cm with a flow rateof 1 ml/min. The column was equilibrated with water [39]. Thesample was eluted with 80% (v/v) methanol and concentratedusing Rotavapor (Buchi Laboratory Equipment, Switzerland). Theconcentrate was further analyzed for vitamin B12 by HPLC followedby derivatization for analysis using ELISA.

HPLC analysis of vitamin B12

All vitamin B12 standards were prepared in double-distilledwater and serially diluted to the concentration range required foranalysis. Vitamin B12 containing samples were filtered through0.45-lm nylon filters and stored refrigerated until analysis. A vol-ume of 10 ll of standard and/or sample was injected into the liquidchromatography device, which had an isocratic condition with amobile phase of 30% methanol in double-distilled water with aset flow rate at 1 ml/min, and analyzed at 361 and 551 nm at28 �C [40]. The retention time of authentic standard of vitaminB12 was recorded. The total chromatographic run was for 10 min.Peak areas of the standards were plotted against the concentrationof vitamin B12, and the resulting standard curve was used to inter-polate vitamin B12 concentrations in the water, pharmaceutical,and food samples.

Cross-reactivityCross-reactivity of the ELISA to a variety of water-soluble vita-

mins was tested, including methylcobalamin, hydroxocobalamin,adenosylcobalamin, vitamin B12 analogs, vitamin B1, vitamin B2,vitamin B5, vitamin B6, vitamin B8, vitamin B9, vitamin C, and vita-min PP. Here 10-fold serial dilution of each vitamin was preparedin distilled water starting at 1000 lg/ml and tested in the assay.The 50% absorbance inhibition (50% B/Bo) and cross-reactivity (%)for each of the compounds were determined.

Studies on interferenceThe effect of various ions or other compounds was studied using

ELISA by fortifying the following solutions from 0.1 to 10,000 ng/ml in distilled water: cobalt, calcium, copper, magnesium, sodium,nitrate, fluoride, phosphate, sulfate, sodium chloride, and hydro-chloric acid. The effect of methanol and acetone on assay perfor-

mance was examined. Samples were tested with the addition of2600 ng/ml derivatized vitamin B12.

Correlation studyA stock of 1000 lg/ml vitamin B12 standard samples was pre-

pared and serially diluted. The samples used for HPLC were 500,1000, 5000, 10,000, 50,000, and 100,000 ng/ml and were preparedin the distilled water. The ELISA samples were serially diluted andderivatized to 10, 50, and 100 ng/ml from the standards used forthe HPLC analysis. Similarly, derivatized vitamin B12 was spikedat a concentration of 100 ng in pharmaceutical and food samplesto check for its accuracy and recovery before and after addition.

Results and discussion

Preparation and characterization of vitamin B12–protein conjugates

Vitamin B12 was coupled to KLH by a mild acid hydrolysis pro-cedure as described in Materials and Methods. As such, vitamin B12

does not have a functional group for conjugation with KLH; there-fore, free carboxyl groups had been created on an unreactive vita-min B12 molecule by mild hydrochloric acid treatment of 6 amidegroups of propionamide side chain of vitamin B12 into mono-, di-,or tricarboxylic vitamin B12 derivative, facilitating the conjugationwith the amine groups of protein as shown in Fig. 1. The vitaminB12–KLH conjugate was characterized by UV spectroscopy. Theabsorbance of vitamin B12–KLH at 361 nm at a concentration of500 mg/L was found to be 0.688. The absorbance of KLH at thesame concentration and wavelength was 0.005; therefore, the dif-ference attributed to vitamin B12 residues is 0.683. Hence, the con-centration of vitamin B12 residues is

0:683=27;500 mol=L ¼ 2:48� 10�5 mol=L;

where 27,500 is the molar extinction coefficient of vitamin B12 at361 nm. Because the residue weight of vitamin B12 is 1300 g permole, its concentration is 2.48 � 10�5 mol/L � 1300 g per mole or32.2 mg/L.

By subtracting the concentration of the vitamin B12 residuesfrom that of vitamin B12–KLH, the concentration (�) molecularweight of KLH is assumed to be 100,000 g and the number of molesper liter of KLH in the sample is

467:8� 10�3 g=L divided by 100;000 g per mole ¼ 4:7� 10�6:

The number of moles of vitamin B12 per mole of KLH is 2.48 � 10�5/4.7 � 10�6 = 5.2.

The conjugation of vitamin B12 carboxylic derivative to KLH bythe EDC and NHS methods at pH 5.5 introduced an average of5.2 mole of derivatized vitamin B12 to each mole of KLH. The differ-ence of hapten density is 5.2 when compared with control KLH,and this is more than that of the previously reported bovine serumalbumin (BSA) conjugate [41]. SDS–PAGE was not possible due toits higher molecular weight (data not shown), but this differencecan be differentiated using HPLC. As such, control KLH eluted outat a retention time of 1.4 and vitamin B12–KLH conjugate elutedout at a retention time of 2.3, as shown in Fig. 2, confirming theconjugation due to an increase in its polar nature of KLH as vitaminB12 is conjugated to KLH.

Characterization of isolated vitamin B12 IgY antibody

The assay specificity was obtained by the use of specific immu-nological reaction between vitamin B12 and vitamin B12 IgY anti-body. Immunizing a white Leghorn hen with vitamin B12–KLHgenerated IgY-class immunoglobulins. Vitamin B12 IgY was ex-tracted from egg yolk by the water dilution method, which was re-

Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246 241

ferred to other extraction procedures in order to obtain high yieldand pure IgY antibody [42]. Purified vitamin B12 IgY antibody wasobtained by KLH affinity column chromatography by exclusion ofany KLH antibody generated as a result of immunization with vita-min B12–KLH. Affinity purification resulted in monospecificity forvitamin B12 antibody toward vitamin B12 with a concentration of2 mg/ml, as shown in Fig. 3. Direct competitive ELISA was testedfor isolated antibodies to obtain better titer values, and it was ob-served that antibodies had higher titer values after the third andfourth booster doses. SDS–PAGE was carried out for further confir-mation of the purity of vitamin B12 IgY antibody. The single bandsin lane 2 in the range of 180–190 kDa suggested the presence of IgYantibody, two distinct bands observed at 60 to 70 kDa and oneband observed at 25 kDa in lane 3 after treatment with b-mercap-toethanol confirmed the presence of heavy chain and light chain,and treatment with pepsin yielding single bands at 45 kDa in lane4 showed the presence of antigen-binding fragment of IgY and sug-gested the absence of any native protein contamination in the puri-fied immunoglobulin samples, as shown in Fig. 4. Using IgY overIgG was advantageous in that bleeding of the animal could beavoided, isolation of antibodies was simple, and less antigen wasrequired to obtain specific IgY with high titer. Moreover, the yieldof IgY antibody was approximately 100 mg/egg yolk without anyIgA or IgM contamination. The possibility of obtaining IgA and

IgM in the egg yolk was very low because both IgA and IgM weretransferred from oviduct into the egg white together with otherproteins. On the other hand, the transfer of IgY in the egg folliclewas receptor mediated; therefore, IgY was reportedly present inegg yolk in large quantities [42].

Standard curve and sensitivity

The antigen-binding capacity of the antibody was estimated bythe direct ELISA checkerboard after purification, as shown in Fig. 5,in which 1:100 dilution of vitamin B12 antibody could give absor-bance around 1.0 at 10 lg/ml vitamin B12–ALP conjugate coatingconcentration. Dose–response data for vitamin B12 calibrators werecollected over 15 days, and 20 replicates were represented; themean standard curve is shown in Fig. 6. At 10 ng/ml, significantinhibition by derivatized vitamin B12 of antibody binding was

Fig.2. HPLC chromatogram of standard KLH (left) and KLH–vitamin B12 conjugate (right).

Fig.3. Elution profile of different fractions of vitamin B12 IgY antibody collectedafter affinity purification through a KLH affinity column and analyzed at 280 nm.

Fig.4. Proteins from each step in the isolation of immunoglobulin from egg yolk(IgY) analyzed on a 12% SDS–polyacrylamide gel run in the presence of 2-mercaptoethanol. Lane 1 (Genei molecular mass markers): myosin, rabbit muscle(205 kDa), phosphorylase b (97.4 kDa), albumin (66 kDa), ovalbumin (43 kDa),carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), and lysozyme(14.3 kDa); lane 2: nonreduced IgY; lane 3: reduced IgY; lane 4: pepsin-treated IgY.

242 Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246

observed. The LOD was 10 ng/ml, and the least detectable dose (LDD)was estimated as 6 ng/ml at 90% B/Bo (mean absorbance value forthe standard divided by mean absorbance value for the zero stan-dard) [43]. The LOD was defined as the lowest derivatized vitaminB12 standard to have a B/Bo value of 10 standard deviation unitsgreater than Bo. The LDD was defined as the lowest derivatizedvitamin B12 standard to have a B/Bo value 3 times greater thanthe standard deviation of Bo [43]. Low-molecular-weight com-pounds such as vitamin B12 are not immunogenic and do not elicitan immune response. To obtain antibodies to such compounds, onemust couple them to a larger molecular-weight molecule (carrier)such as KLH, as shown in Fig. 1. The binding site of antibodiesraised against the low-molecular-weight compound (hapten) con-jugated to the larger molecular-weight carrier may also includerecognition of the chemical linker. One way to increase the affinityof an antibody for a low-molecular-weight compound is to convertthe hapten in the sample into a derivative that mimics the immu-nogen by the chemical addition of a reagent identical or similar tothe spacer or chemical linker [44]. Derivatization of analytes hasbeen shown to increase immunoassay sensitivity [44–46]. Deriva-tization of vitamin B12 in samples with hydrochloric acid allowedus to increase the sensitivity of the assay by approximately 10-fold,from 100 to 10 ng/ml, as shown in Fig. 6.

Affinity of a purified vitamin B12 IgY antibody

Affinity of the vitamin B12 IgY antibody for derivatized vitaminB12 was calculated from the dose–response curve of the competi-

tive ELISA by applying the Scatchard analysis equation mentionedin Materials and Methods. The affinity constant (Ka) was calculatedto be 4.23 � 108 L/mol, as shown in Fig. 7. The obtained affinity va-lue implies high binding of antibody with the derivatized vitaminB12.

Specificity

In determining the specificity of the assay, it is important toinvestigate structurally related compounds to inhibit vitamin B12

antibody binding. The cross-reactivity data are summarized inTable 1. The assay is more specific for derivatized vitamin B12

and less specific for other derivatives, and there is virtually nocross-reactivity (<0.01%) against the water-soluble vitamincompounds. This shows that vitamin B12 must be derivatized toincrease its sensitivity before its analysis using ELISA. The standardcurves of other forms of vitamin B12 to affinity-purified vitaminB12 IgY antibody are shown in Fig. 8 and have sensitivities ofapproximately 100 ng/ml.

Matrix effects

The effects on the immunoassay of various inorganic and organ-ic contaminants often found in samples were determined by add-ing them to distilled water (Table 2). The assay is not affected bymethanol or acetone at concentrations as high as 5%. The presence

Fig.6. Standard curve for derivatized vitamin B12 and vitamin B12. Each pointrepresents the mean of 20 determinations. Vertical bars indicate error bars with 5%value.

Table 1Cross-reactivity of ELISA to various forms of water-soluble vitamin.

Forms of vitamin B12 50% B/Bo (ng/ml) Cross-reactivity (%)

Derivatized vitamin B12 2600 100.0Vitamin B12 7500 34.6Methylcobalamin 9400 27.6Hydroxocobalamin 17,700 14.6Adenosylcobalamin 6800 38.2Analogs of vitamin B12 21,300 12.2Vitamin B1 (thiamine) NR <0.01Vitamin B2 (riboflavin) NR <0.01Vitamin B5 (pantothenic acid) NR <0.01Vitamin B6 (pyridoxine) NR <0.01Vitamin B8 (biotin) NR <0.01Vitamin B9 (folic acid) NR <0.01Vitamin C (ascorbic acid) NR <0.01Vitamin PP (niacin) NR <0.01

Note: NR, no response.

Fig.5. Checkerboard ELISA of antibody obtained after purification.

Fig.7. Scatchard plot for vitamin B12 IgY antibody showing its affinity towardderivatized vitamin B12.

Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246 243

of cobalt, calcium, copper, magnesium, nitrate, and sodium fluorideat 10,000 ng/ml and of phosphate and sulfate at 100 ng/ml also hadno effect on the vitamin B12 immunoassay. Hydrochloric acidabove 0.25 N caused some interference, indicating that samplespreserved with acid should be neutralized prior to evaluation ofsamples using the ELISA.

Precision

Water samples were spiked with vitamin B12 and derivatized atfour different concentrations for precision studies. Each was mea-sured 5 times in duplicate on 5 different days (Table 3). The coef-

ficients of variation were between 0.002% and 1.2% for bothinter- and intraassays. However, in the case of food samples, vita-min B12 was extracted and derivatized for precision studies.

Accuracy

Accuracy was assessed by analyzing the pharmaceutical andfood samples before and after the addition of derivatized vitaminB12. On average, 99% to 101% of added derivatized vitamin B12

was recovered, as shown in Table 4.

Comparison study

Four different vitamin B12 samples were compared using theELISA and HPLC. The results obtained by the ELISA and HPLC agreedwell with the labeled values for vitamin B12 injection, tablets,

Table 3Results after water samples were spiked with vitamin B12 and derivatized at fourdifferent concentrations for precision studies using direct ELISA.

10 ng/ml

100 ng/ml

1000 ng/ml

10,000 ng/ml

Replicates/day 5 5 5 5Days 5 5 5 5n 25 25 25 25Mean (ng/ml) 8.5 93.4 994.6 9992.6Standard deviation 1.05 3.44 4.32 5.32Relative standard

deviation12.33 3.67 0.43 0.05

Recovery (%) 85.0 93.4 99.4 99.0% CV (intraassay) 1.23 0.04 0.004 0.0005% CV (interassay) 1.03 0.06 0.03 0.009

Note: Samples were assayed in replicates of 5 assays per day over 5 days. CV,coefficient of variation.

Table 4Results after recovery of externally added (100 ng/ml) derivatized vitamin B12 inpharmaceutical and multivitamin chocolate was analyzed using ELISA method.

Pharmaceutical samplesand multivitaminchocolate

Added(ng/ml)

Found(ng/ml)

Relativestandarddeviation (%)

Recovery(%)

Injections 0 94.6 4.82 99.1100 193.0 3.00

Tablets 0 91.4 3.51 100.1100 191.6 1.86

Capsules 0 93.6 8.26 99.3100 192.4 2.09

Commercial multivitaminchocolates

0 84.0 7.08 101.5100 186.8 3.77

Note: Data were derived from replicate assays (n = 3).

Table 5Analysis of vitamin B12 in pharmaceutical and multivitamin chocolate using ELISA incomparison with HPLC.

Pharmaceutical and foodsamples

Amountlabeled

Amount found

ELISA HPLC

Multivitamin injection 0.334 0.29 ± 0.03 0.30 ± 0.01Multivitamin tablet 15 14.12 ± 0.23 14.74 ± 0.23Multivitamin capsule 15 14.38 ± 0.44 14.98 ± 0.14Commercial multivitamin

chocolate0.5 0.39 ± 0.02 0.41 ± 0.02

Note: The averages of five measurements ±standard deviations are shown. Theamount of vitamin B12 labeled in multivitamin injections is 0.334 mg/ml, a multi-vitamin tablet is 15 lg/tablet, a multivitamin capsule is 15 lg/tablet, and a multi-vitamin chocolate is 0.5 lg/100 g.

Fig.9. Linear regression graph of correlation between ELISA and HPLC.

Fig.8. Standard curve for other forms of vitamin B12. Each point represents themean of 20 determinations. Vertical bars indicate errors bars with 5% value.

Table 2Effect of various ions and organic compounds on ELISA for vitamin B12.

Compound Concentration (ng/ml) Recovery (%)

Cobalt chloride 10,000 98.0Calcium chloride 10,000 90.8Copper chloride 5000 96.0Magnesium chloride 10,000 94.4Sodium chloride 10,000 94.2Calcium sulfate 100 94.4Magnesium sulfate 100 100.0Sodium fluoride 10,000 93.6Sodium nitrate 10,000 98.4Sodium phosphate 100 93.2Acetone <5% 93.6Methanol <5% 99.2Hydrochloric acid 0.5 N 77.2

244 Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246

capsules, and multivitamin chocolates, as shown in Table 5. Acomparison of two different analytical techniques, HPLC and ELISA,demonstrates the usefulness of the vitamin B12 immunoassay inFig. 9. There was no difference between the two methods, as deter-mined. To compare the results of the two different analytical meth-ods (ELISA vs. HPLC) at different concentration ranges, the ELISAconcentration values were well correlated to fall within the detec-tion range of the HPLC method.

Conclusion

A competitive direct ELISA for derivatized vitamin B12 has beendeveloped and is a useful and effective tool that will enable theanalysis of vitamin B12 in selected food samples economically.The tested HPLC method used for comparison is an HPLC–UVmethod. UV detectors are not as sensitive as others available onthe market (e.g., tandem mass spectrometry [MS/MS] devices).Therefore, the statement is true for the instrumentation setupused; however, a Waters application note about an ultra-perfor-mance liquid chromatography (UPLC)–MS/MS analysis of water-soluble vitamins shows values for B12 of 0.0024 ng/ll (= 2.4 mg/ml, infant formula) and 0.0039 ng/ll (= 3.9 ng/ml, dietary supple-ment vitamin tablet). Those values are below the working rangeof the presented ELISA method (LOD = 10 ng/ml). Therefore, ELISAis more sensitive than HPLC to have a lower detection limit. Fur-thermore, potential matrix effects from food samples after extrac-tion had little or no effect on the ELISA result when compared withthe HPLC method. Application of IgY in analytical and in vitro stud-ies is advantageous when compared with IgG in terms of its stabil-ity, high antibody production capacity, and cost effectiveness. Themethod offers reliable results and can be adopted as a routine ana-lytical method for the determination of trace vitamin B12 in biolog-ical samples.

Acknowledgments

We thank the director of the Central Food Technological Re-search Institute (Mysore, India) for providing constant encourage-ment. L. Sagaya Selva Kumar is grateful to the Council of Scientificand Industrial Research for providing a Senior Research Fellowship.

References

[1] V. Herbert, Vitamin B12, in: E.E. Ziegler, L.J. Filer (Eds.), Present Knowledge inNutrition, seventh ed., ILSI Press, Washington, DC, 1996, pp. 191–205.

[2] M.A. Bernard, P.A. Nakonezny, T.M. Kashner, The effect of vitamin B12

deficiency on older veterans and its relationship to health, J. Am. Geriatr.Soc. 46 (1998) 1199–1203.

[3] Food and Nutrition Board, Institute of Medicine, Dietary Reference Intakes forThiamine, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid,Biotin, and Choline, National Academy Press, Washington, DC, 2000. <http://www.nap.edu/catalog/6015.html>.

[4] H. Wolfgang, G. Jurgen, Vegetarian lifestyle and monitoring of vitamin B12

status, Clin. Chim. Acta 326 (2002) 47–59.[5] Food and Nutrition Board, Institute of Medicine, Dietary Reference Intakes:

Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid,Biotin, and Choline, National Academy Press, Washington, DC, 1998.

[6] Z. Schneider, A. Stroinski, Comprehensive B12, Walter de Gruyter, New York,1987. pp. 111–115.

[7] S.T. Hodson, S. Subramanian, J.R. Allen, Determination of pantothenic acid,biotin, and vitamin B12 in nutritional products, J. Assoc. Off. Anal. Chem. 67(1984) 994–998.

[8] R.C. Oh, L.D. Brown, Vitamin B12 deficiency, Am. Fam. Physician 67 (2003) 979–986.

[9] A. Kumudha, S.S. Kumar, M.S. Thakur, G.A. Ravishankar, R. Sarada, Purification,identification, and characterization of methylcobalamin from Spirulinaplatensis, J. Agric. Food Chem. 58 (2010) 9925–9930.

[10] B.P. Kelleher, J.M. Scott, S.D. O’Broin, Cryopreservation of Lactobacillusleichmannii for vitamin B12 microbiological assay, Med. Lab. Sci. 47 (1990)90–96.

[11] P.J. Casey, K.R. Speckman, F. Ebert, W.E. Hobbs, Radioisotope dilutiontechnique for determination of vitamin B12 in foods, J. Assoc. Off. Anal.Chem. 65 (1982) 85–88.

[12] K. Muhammed, D. Briggs, G. Jones, Comparison of a competitive binding assaywith Lactobacillus leichmannii A.T.C.C. 7830 assay for the determination ofvitamin B12 in foods, Food Chem. 48 (1993) 431–434.

[13] H.B. Li, F. Chen, Y. Jiang, Determination of vitamin B12 in multivitamin tabletsand fermentation medium by high-performance liquid chromatography withfluorescence detection, J. Chromatogr. A 891 (2000) 243–247.

[14] C.K. Markopoulou, K.A. Kagkadis, J.E. Koundourellis, An optimized method forthe simultaneous determination of vitamins B1, B6, B12, in multivitamin tabletsby high performance liquid chromatography, J. Pharm. Biomed. Anal. 30(2002) 1403–1410.

[15] P. Moreno, V. Salvado, Determination of eight water- and fat-soluble vitaminsin multi-vitamin pharmaceutical formulations by high-performance liquidchromatography, J. Chromatogr. A 870 (2000) 207–215.

[16] P. Vinas, N. Campillo Lopez-Garcia, I.M. Hernandez-Cordoba, Speciation ofvitamin B12 analogues by liquid chromatography with flame atomicabsorption spectrometric detection, Anal. Chim. Acta 318 (1996) 319–325.

[17] M. Morita, T. Uehiro, K. Fuwa, Comments on the critical evaluation of curvefitting in infrared spectrometry, Anal. Chem. 52 (1980) 351–352.

[18] H. Karsilayan, Quantitation of vitamin B12 by first-derivative absorptionspectroscopy, Spectrochim. Acta 52 (1996) 1163–1168.

[19] B. Morelli, High-resolution/higher-order derivative spectrophotometry fordetermination of ternary mixtures of B-complex vitamins inpharmaceuticals, Fresenius J. Anal. Chem. 354 (1996) 97–102.

[20] S.A. Baker, Determination of cobalamins using capillary electrophoresisinductively coupled plasma mass spectrometry, Spectrochim. Acta B 55(2000) 1823–1832.

[21] S.S. Kumar, R.S. Chouhan, M.S. Thakur, Enhancement of chemiluminescence forvitamin B12 analysis, Anal. Biochem. 388 (2009) 312–316.

[22] R. Sharma, Y.S. Rajput, G. Dogra, S.K. Tomar, Estimation of vitamin B12 by ELISAand its status in milk, Milchwiwissenschaft 62 (2007) 127–130.

[23] E.M. Akita, S. Nakai, Comparison of four purification methods for theproduction of immunoglobulins from eggs laid by hens immunized with anenterotoxigenic E. coli strain, J. Immunol. Methods 160 (1993) 207–214.

[24] P.H. Fortgens, C. Dennison, E. Elliott, Anti-cathepsin D chicken IgY antibodies:characterisation, cross-species reactivity, and application to immunogoldlabelling studies of human neutrophils and other tissues,Immunopharmacology 36 (1997) 305–311.

[25] G. Camenisch, M. Tini, D. Chilov, I. Kvietikova, V. Srinivas, J. Caro, P. Spielmann,R.H. Wenger, General applicability of chicken egg yolk antibodies: theperformance of IgY immunoglobulins raised against the hypoxia-induciblefactor 1a, FASEB J. 13 (1999) 81–88.

[26] M. Tini, U.R. Jewell, G. Camenisch, D. Chilov, M. Gassmann, Generation andapplication of chicken egg-yolk antibodies, Comp. Biochem. Physiol. A 131(2002) 569–574.

[27] P.M. Finglas, M.R.A. Morgan, The determination of vitamins in food bybiospecific analysis, Int. Ind. Biochem. 8 (1988) 9–12.

[28] M.R.A. Morgan, New techniques in food analysis: immunoassays and theirapplication to small molecules, J. Assoc. Public Anal. 23 (1985) 59–63.

[29] C.D. Tsalta, S.A. Rosario, G.S. Cha, L.G. Bachas, M.E. Meyerhoff, A solid-phase enzyme-linked assay for vitamin B12, Mikrochim. Acta 1 (1989) 65–74.

[30] M.I. Watkins, C.R. Barlett, E.T.W. Lian, J.M. Pocekay, M.A. Staples, B12 enzymeimmunoassay and sample pretreatment, U.S. Patent 5,187,107, 1992.

[31] L.D. Anton, P.C.H. Hogenkamp, E.T. Walker, A.N. Matwiyoff, Carbon-13 nuclearmagnetic resonance studies of the monocarboxylic acids of cyanocobalamin:assignments of the b-, d-, and e-monocarboxylic acids, J. Am. Chem. Soc. 102(1980) 2215–2219.

[32] F.B. Erlanger, F. Borek, M.S. Beiser, S. Lieberman, Steroid–protein conjugates: I.Preparation and characterization of conjugates of bovine serum albumin withtestosterone and with cortisone, J. Biol. Chem. 228 (1957) 713–728.

[33] M. Haak-Frendscho, Eggstract why IgY? Chicken polyclonal antibody, anappealing alternative, Promega Notes 46 (1995) 11–17.

[34] G.T. Hermanson, A.K. Mallia, P.K. Smith, Immobilized Affinity LigandTechniques, Academic Press, San Diego, 1992.

[35] R.S. Chouhan, A.C. Vinayaka, M.S. Thakur, Thiol-stabilized luminescent CdTequantum dot as biological fluorescent probe for sensitive detection of methylparathion by a fluoro-immunochromatographic technique, Anal. Bioanal.Chem. 397 (2010) 1467–1475.

[36] U.K. Laemmli, Cleavage of structural proteins during the assembly of the headof bacteriophage T4, Nature 227 (1970) 680–685.

[37] E.M. Akita, S. Nakai, Production and purification of Fab’ fragments fromchicken egg yolk immunoglobulin Y (IgY), J. Immunol. Methods 162 (1993)155–164.

[38] H. Feldman, D. Rodbard, Mathematical theory of radioimmunoassay, in: W.D.Odell, W.M. Daughaday (Eds.), Principles of Competitive Protein BindingAssay, Springer, Philadelphia, 1971, pp. 158–171.

[39] W.A. Fenton, L.E. Rosenberg, Improved techniques for the extraction andchromatography of cobalamins, Anal. Biochem. 90 (1977) 119–125.

[40] H.-B. Li, F. Chen, Y. Jiang, Determination of vitamin B12 in multivitamin tabletsand fermentation medium by high-performance liquid chromatography withfluorescence detection, J. Chromatogr. A 891 (2000) 243–247.

[41] S.S. Ahrenstedt, I.J. Throrell, The production of antibodies to vitamin B12, Clin.Chim. Acta 95 (1979) 419–423.

[42] E.M. Akita, E.C.Y. Li-Chan, Isolation of bovine immunoglobulin G subclassesfrom milk, colostrum, and whey using immobilized egg yolk antibodies, J.Dairy Sci. 81 (1998) 54–63.

Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246 245

[43] A.R. Midgley, G.D. Niswender, R.W. Rebar, Principles for the assessment ofreliability of radioimmunoassay methods (precision, accuracy, sensitivity,specificity), Acta Endocrinol. 63 (1969) 163–179.

[44] G. Kertesz, B. Bourcier, H. Cailla, F. Jean, Immunoradiometric assay ofsuccinylated corticotropin: an improved method for quantification of ACTH,Clin. Chem. 44 (1998) 78–85.

[45] R. Yamane, T. Nakamura, E. Matsuura, H. Ishige, M. Fujimoto, A simple andsensitive radioimmunoassay for adenosine, J. Immunoassay 12 (1991) 501–519.

[46] X.L. Qing, B.D. Hammock, J.M. Seiber, Development of an enzyme-linkedimmunosorbent assay for the herbicide bentazon, J. Agric. Food Chem. 39(1991) 1537–1544.

246 Competitive immunoassay for vitamin B12 / L.S. Selva Kumar, M.S. Thakur / Anal. Biochem. 418 (2011) 238–246

Analytica Chimica Acta 722 (2012) 107– 113

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

j ourna l ho me page: www.elsev ier .com/ locate /aca

Dipstick based immunochemiluminescence biosensor for the analysis of vitaminB12 in energy drinks: A novel approach

L.S. Selvakumar, M.S. Thakur ∗

Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (a constituent laboratory of the Council of Scientific and Industrial Research,CSIR), Mysore 570020, Karnataka, India

a r t i c l e i n f o

Article history:Received 9 November 2011Received in revised form 11 January 2012Accepted 5 February 2012Available online 13 February 2012

Keywords:BiosensorDipstickImmunochemiluminescenceEnzyme linked immunosorbent assayVitamin B12

a b s t r a c t

In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for thedetection of vitamin B12 in energy drinks. The method is a direct competitive type format involvingthe immobilization of vitamin B12 antibody on nitrocellulose membrane (NC) followed by treat-ment with vitamin B12 and vitamin B12–alkaline phosphatase conjugate to facilitate the competitivebinding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generatechemiluminescence (CL). The number of photons generated was inversely proportional to the vitaminB12 concentration. After systematic optimization, the limit of detection was 1 ng mL−1. The coefficientof variation was below 0.2% for both intra- and inter-assay precision. Vitamin B12 was extracted fromenergy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were ana-lyzed, and a good correlation was observed when the data were compared with a reference enzyme linkedimmuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, andhigh-throughput screening of vitamin B12 in energy drinks samples. The dipstick technique based onimmuno-CL is suitable for the detection of several analyte in food and environmental samples.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

An immunological method is one of the promising tools for thedevelopment of easy-handling biosensors. Besides, immunoassaymethods are sensitive, cost-effective, easy to perform, and requirea small sample volume [1]. However, such techniques often requirelong reaction times and involve multiple steps [2]. The utilizationof these immunoassays has been confined to laboratories equippedwith tools and devices for analysis. Therefore, the convenience andspeed of the test have been achieved by a novel concept of immun-odipstick that depends on the transportation of a reactant to itsbinding partner immobilized on a membrane surface [3]. It com-bines several benefits including a user-friendly format, short assaytime, long-term stability over a wide range of climates, and cost-effectiveness. These characteristics make it ideally suited for on-sitescreening by people who are not skilled analysts [4].

Vitamin B12 is one such analyte needs urgency for the detection.Vitamin B12 belongs to the B vitamin group and prevents perniciousanemia, which is caused by vitamin B12 deficiency. Vegetarians andpeople who do not eat meat need to supplement their diet by tak-ing multivitamin tablets and beverages supplemented with vitamin

∗ Corresponding author. Tel.: +91 821 2515792; fax: +91 821 2517233.E-mail addresses: msthakur@cftri.res.in, msthakur@yahoo.com (M.S. Thakur).

B12 as plant products contain very little amount of vitamin B12.As the excessive consumption of vitamin B12 may cause asthmaand folic acid deficiency, therefore, typically only a low level ofvitamin B12 (e.g., ng g−1) is added to products, thus making directanalysis difficult. The most common requirement for the analysisof vitamin B12 is in the quality control of pharmaceuticals (tabletsor injections), blood plasma serum, milk products for infants, andfermentation products which involves complicated sample prepa-ration [5]. The daily requirements of vitamin B12 are very low whencompared with other vitamins [6], and deficiencies are reported tobe at the nanogram level [7]. Extracting vitamin B12 from a largeramount of sample is simple and effective for some relatively largeand solid samples, such as Algae [8]. However, this strategy is notalways suitable for liquid samples (e.g., beverages). At the onset ofthese challenges, it is very important to diagnose vitamin B12 at asensitive level.

The conventional methods for the detection of this compoundfor analytical purposes are including a microbiological assay thatuses Lactobacillus leichmannii as a test organism is very laborious[9]. Radioisotope method has been applied to the determination ofvitamin B12 in food samples using intrinsic factor as recognitionmolecule but the cost of the assay is questionable [10,11]. Highperformance liquid chromatography (HPLC) has been employed bya number of workers to assay vitamin B12 in multivitamin andmineral tablets where as these matrices are less complex than

0003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2012.02.006

108 L.S. Selvakumar, M.S. Thakur / Analytica Chimica Acta 722 (2012) 107– 113

foodstuffs and therefore, extraction and resolution of the vitaminis quite difficult in food matrix [12,13]. Other specific detectionmethods based on the dosage of cobalt either by atomic absorptionspectrometry [14] or by inductively coupled argon plasma emis-sion spectrometric detection [15] were proposed. However, thelimit of detection (LOD) of these methods (150 mg g−1) only allowsthe quantification of high amounts of vitamin B12 in food samples.Spectrophotometry [16,17], fluorimetric assay [18], capillary elec-trophoresis [19], and CL [20] are employed for analysis of vitaminB12. However these methods are found to be less dependable inanalyzing crude sample considering the time, cost and sensitivity.Such a situation demands more specific methods like biospecificassays which make use of biorecognition molecules like antibodies,for the detection of analyte [21]. Development of an immuno-logical based methods, is therefore a promising field; taken care,the possibilities of exploiting the principle of antigen–antibodyspecific interaction. But the issues of raising antibody, IgG andmonoclonal antibody against such analyte is always question-able due to its costly extraction procedure which is resolved withthe development of efficient protocols for raising and extractingimmunoglobulins from hen egg yolk, namely IgY [22]. In our previ-ous report, we used hens as immunization hosts to produce vitaminB12 IgY antibody against a derived form of vitamin B12 by immuniz-ing a hen with vitamin B12 conjugated to a carrier protein, KeyholeLimpet Hemocyanin. The sensitivity and specificity of developedantibodies were checked using ELISA [23]. Previous studies in ourlaboratory indicated that, IgY antibody in combination with CL washighly promising for detection of analytes with high sensitivity[24].

In present work, we advance the approach of using dip-stick based format of immuno-CL method for the detection ofvitamin B12. CL-based analytical methods are rapid, specific,cost-effective, and requires no excitation source like in fluores-cence, phosphorescence, monochromator (often not even a filter),or radioactive or hazardous chemicals. Hence, this method hasbecome an attractive analytical tool for sensitive clinical diag-nosis and environmental applications [25–27]. To achieve thisobjective, different parameters were optimized such as immobi-lization of vitamin B12 antibodies IgY on NC strip, optimization ofvitamin B12–ALP (alkaline phosphatase) conjugate concentration,optimization of substrate CDP star concentration, optimization ofvolume of substrate CDP star concentration along with possiblemechanism of chemiluminescence reaction. The CL signal pro-duced by biochemical reactions was indirectly proportional to theconcentration of vitamin B12. In addition, the CL analytical proce-dure was compared and validated with conventional colorimetricassays.

2. Materials and methods

2.1. Reagents

All the reagents were analytical grade and used withoutfurther purification. Doubly distilled water (DDW) was usedthroughout this work. Vitamin B12, Alkaline phosphatase,Tween-20, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) cabodiimide (EDC), XAD-2 Amberlite,Disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro) tricycle [3.3.1.13,7]decan}-4-yl) phenyl phosphate (CDPstar) chemilumeniscent substrate were procured from M s−1

Sigma–Aldrich Chemicals, USA. Energy drinks were procured fromlocal market, Mysore, India. Vitamin B12 antibody generation andvitamin B12–alkaline phosphatase conjugate preparation weredone at Central Food Technological Research Institute (CFTRI),India.

2.2. Apparatus

Luminometer-Luminoskan TL Plus with photomultiplier tubewas procured from Helsinki, Finland. Incubator shaker-INFORS HT,Ecotron was procured from Bottmengen, Switzerland. Vortex cyclomixer was procured from Genie Bangalore, India. Flash evaporatorwas procured from Rotavac Senso Heidolph, Sweden. Nitrocellulosemembrane was procured from Advanced Microdevices, AmbalaCantt Haryana, India.

2.3. Experimental set up and principle of detection of vitamin B12using dipstick based immuno-CL method

The principle is competitive binding between the immobi-lized vitamin B12 antibodies with free vitamin B12 and vitaminB12–ALP conjugated on NC membrane. The CL signal generatedduring reaction was inversely proportional to the presence of vita-min B12 in the samples. Thereby, the concentration of vitaminB12 could be determined by measuring the CL intensity in theabsence and presence of vitamin B12 as shown in Scheme 1a. TheCL signals were plotted at 5 s intervals for a period of 10 min. TheCL depends upon the light signals generated by the biochemi-cal reactions between vitamin B12–ALP and CDP-star as shown inScheme 1b. Enzymatic dephosphorylation of dioxetane by alkalinephosphatase leads to the formation of the meta-stable dioxetanephenolate anion, which decomposes and emits light and is detectedby the luminometer. The CL units (CLU) term used for the unitvalue of photons produced from the CL reaction between vita-min B12–ALP and CDP-star over fixed time duration. Using thisphenomenon, vitamin B12 concentration in analytical samples wasdetermined.

2.4. Preparation of hapten–protein conjugates containingvitamin B12 epitopes and generation of vitamin B12 IgY antibodies

Preparation of hapten–protein conjugate containing vitaminB12 epitopes and production of the IgY polyclonal antibod-ies against vitamin B12 is described in our recent publication[23].

2.5. Competitive direct ELISA for vitamin B12 determination

A microtiter plate was coated with 100 ng well−1 of the puri-fied antibody with 50 mmol L−1 of Na2CO3–NaHCO3 solution (pH9.6) and incubated overnight at 4 ◦C. After the coating solutionwas removed, blocking solution (200 �L well−1 of 1% gelatin inphosphate buffer saline (PBS), containing 0.1% NaN3) was added,and the wells were washed with phosphate buffer saline tween(PBST) after 2 h incubation at 37 ◦C. The plate was then filled with100 �L well−1 of different concentration of derivatized vitaminB12 sample, along with 100 �L well−1 of serially diluted vitaminB12–ALP conjugates, and incubated for 1 h at 37 ◦C. The wells werethen washed thrice using 200 �L PBST, and color development wasdone using p-nitrophenyl phosphate (1 mg mL−1; 100 �L well−1) in1% diethanolamine buffer, pH 9.8, at 37 ◦C for 30 min. The reac-tion was stopped by adding 3 M NaOH (40 �L well−1), and theabsorbance was read at 405 nm in an ELISA microplate reader. Allanalysis was done in triplicates.

2.6. Immobilization of vitamin B12 antibodies IgY on NC strip

Different concentrations of antibodies were immobilized, from1, 10, 100, 1000 ng �L−1 by direct spotting on to the surface ofNC membrane and dried at room temperature (RT) for 60 min.Blocking was done with 2% skimmed milk in PBS for 30 min inshaking condition. Strips were washed several times with PBS

L.S. Selvakumar, M.S. Thakur / Analytica Chimica Acta 722 (2012) 107– 113 109

Scheme 1. (a) Schematic representation of immuno-CL based dipstick technique for detection of vitamin B12. (b) Enzymatic dephosphorylation of dioxetane by alkalinephosphatase.

Adopted from https://e-labdoc.roche.com/LFR PublicDocs/ras/11759051001 en 08.pdf.

then dipped in optimized concentration of vitamin B12–ALP con-jugate. After 60 min of incubation the strips were again washedwith PBS (pH 7.4) and dried. The CL of the strip was checkedusing optimized concentration/volume of CDP star in a lumi-nometer. The photons generated as a result of the reaction wasrecorded for a period of 10 min using the luminometer andconverted into a readable format as CLU using hexa terminal soft-ware.

2.7. Optimization of vitamin B12–ALP conjugate concentration

NC membrane strips were coated with optimized concentra-tions of antibodies and dried at RT for 60 min. Blocking was donewith 2% skimmed milk in PBS for 30 min in shaking condition.Strips were washed several times with PBS then dipped in differ-ent concentration of vitamin B12–ALP conjugate from 6.25, 12.5,25, 50, 100 �g mL−1. After 60 min of incubation the strips wereagain washed with PBS (pH 7.4) and dried. The CL of the stripwas checked using optimized concentration/volume of CDP starin a luminometer. The photons generated as a result of the reac-tion was recorded for a period of 10 min using the luminometer

and converted into a readable format as CLU using hexa terminalsoftware.

2.8. Optimization of substrate CDP star concentration

NC membrane containing strips were coated with optimizedconcentrations of antibodies and dried at RT for 60 min. Blockingwas done with 2% skimmed milk in PBS for 30 min in shaking con-dition. Strips were washed several times with PBS then, dippedin optimized concentration of vitamin B12–ALP conjugate. After60 min of incubation the strips were again washed with PBS (pH 7.4)and dried. The CL of the strip was checked using different concentra-tion of CDP star from 3.125, 6.25, 12.5 and 25 �M in a luminometer.The photons generated as a result of the reaction were recorded fora period of 10 min using the luminometer and converted into areadable format as CLU using hexa terminal software.

2.9. Optimization of volume of substrate CDP star concentration

NC membrane containing strips were coated with optimizedconcentrations of antibodies and dried at RT for 60 min. Block-ing was done with 2% skimmed milk in PBS for 30 min in shaking

110 L.S. Selvakumar, M.S. Thakur / Analytica Chimica Acta 722 (2012) 107– 113

condition. Strips were washed several times with PBS then, dippedin optimized concentration of vitamin B12–ALP conjugate. After60 min of incubation the strips were again washed with PBS (pH7.4) and dried. The CL of the strip was checked using optimized con-centration of CDP star varying volume from 100, 200, 400, 800 �Lin a luminometer. The photons generated as a result of the reac-tion were recorded for a period of 10 min using the luminometerand converted into a readable format as CLU using hexa terminalsoftware.

2.10. Detection of vitamin B12 by dipstick based immuno-CL assay

NC membrane containing strips was coated with optimized con-centrations of antibodies and dried at RT for 60 min. Blocking wasdone with 2% skimmed milk in PBS for 30 min in shaking condition.Strips were washed several times with PBS and finally dipped in dif-ferent concentration of derivatized vitamin B12 from 1, 10, 100 and500 ng mL−1 followed by 60 min of incubation in optimized concen-tration of vitamin B12–ALP conjugate and dried. The CL of the stripwas checked using optimized concentrations/volume of CDP star.The photons generated as a result of the reaction were recordedfor a period of 10 min using the luminometer and converted into areadable format using as CLU hexa terminal software.

2.11. Extraction of vitamin B12 from energy drinks and itscorrelation study

200 mL of energy drinks having vitamin B12 labeled at concen-tration of 2 �g 100 mL−1 was extracted using cationic resin XAD-2Amberlite [8]. The sample was loaded onto Amberlite XAD-2, pre-pared as a methanolic suspension of the resin packed to a bed heightof 15–16 cm. The column was equilibrated with water. The sam-ple was eluted with 80% (v/v) methanol and concentrated usingrotavapor (Buchi). The concentrate was derivatized and diluted forfurther analysis of vitamin B12 by ELISA followed by analysis usingdipstick based immuno-CL. Energy drinks was further spiked with10 ng of concentration of derivatized vitamin B12 for its accuracyand recovery before and after addition.

3. Results and discussion

3.1. Effect of antibody concentration for immobilization

Different concentrations of vitamin B12 antibody were immo-bilized by direct spotting on NC membrane to see the optimumbinding of antibody with vitamin B12-conjugate. Out of which100 ng �L−1 was found to be optimum because beyond 100 ng �L−1

CL signals obtained were almost constant which shows thesaturation binding of antibody and antigen reaction therefore,1000 ng �L−1 was ruled out. Comparatively lower CL signals wereobtained with lower antibody concentration due to less binding ofantibody with vitamin B12-conjugate as shown in Fig. 1.

3.2. Effect of vitamin B12–ALP conjugate concentration

Optimized immobilized concentrations of vitamin B12 antibodywere dipped in different concentration of vitamin B12–ALP conju-gate to check for optimum binding of vitamin B12–ALP conjugateto vitamin B12 antibody. Out of which, 50 �g mL−1 was found tobe optimum because beyond 50 �g mL−1 CL signals obtained werealmost constant which shows the saturation binding of antibodyand antigen reaction therefore, 100 �g mL−1 was ruled out. Com-paratively lower CL signals were obtained with lower vitaminB12–ALP conjugate concentration due to less binding of vitaminB12–conjugate to vitamin B12 antibody as shown in Fig. 2.

Fig. 1. Effect of antibody for immobilization. The reaction mixtures contained dif-ferent concentrations of antibody from 1, 10, 100, 1000 ng �L−1 by direct spottingon to the surface of NC membrane followed by blocking with 2% skimmed milk.Strips were dipped in 50 �g mL−1 concentration of vitamin B12–ALP conjugate tocheck for optimum CL generation using 12.5 �M (400 �L) concentration CDP starin a luminometer. The photons generated as a result of the reaction were recordedfor a period of 10 min using the luminometer and converted into a readable formatusing hexa terminal software as CLU. Data were derived from triplicate assays (n = 3)with error bars of 5%.

3.3. Effect of substrate CDP star concentration

Optimized concentrations of vitamin B12 antibody/vitaminB12–ALP conjugate was dipped in different concentration of ALPspecific CDP star substrate to check for optimum CL signals. Out ofwhich, 12.5 �M concentration was found to be optimum becausebeyond 12.5 �M concentration substrate availability to enzyme ismore results in higher undesirable background CL signals there-fore 25 �M concentration was ruled out. Comparatively lower CLsignals were obtained with lower CDP Star concentration due to

Fig. 2. Effect of vitamin B12–ALP conjugate concentration. The reaction mixturescontained antibody of 100 ng �L−1 by direct spotting on to the surface of NC mem-brane followed by blocking with 2% skimmed milk. Strips were dipped in differentconcentration of vitamin B12–ALP conjugate from 6.25, 12.5, 25, 50, 100 �g mL−1 tocheck for optimum CL generation using 12.5 �M (400 �L) concentration CDP starin a luminometer. The photons generated as a result of the reaction were recordedfor a period of 10 min using the luminometer and converted into a readable formatusing hexa terminal software as CLU. Data were derived from triplicate assays (n = 3)with error bars of 5%.

L.S. Selvakumar, M.S. Thakur / Analytica Chimica Acta 722 (2012) 107– 113 111

Fig. 3. Effect of substrate CDP star concentration. The reaction mixtures containedantibody of 100 ng �L−1 by direct spotting on to the surface of NC membranefollowed by blocking with 2% skimmed milk. Strips were dipped in 50 �g mL−1 con-centration of vitamin B12–ALP conjugate to check for optimum CL generation usingoptimized volume of different concentration of CDP star from 3.125, 6.25, 12.5 and25 �M in a luminometer. The photons generated as a result of the reaction wererecorded for a period of 10 min using the luminometer and converted into a read-able format using hexa terminal software as CLU. Data were derived from triplicateassays (n = 3) with error bars of 5%.

less substrate available for enzyme to generate CL signals as shownin Fig. 3.

3.4. Effect of substrate CDP star volume

Optimized concentrations of vitamin B12 antibody/vitaminB12–ALP conjugate were dipped in different volume of optimizedconcentration of ALP specific CDP star substrate in cuvette to checkfor optimum CL signals and minimum volume require for dipstickto immerse in substrate solution. Out of which, 400 �L was found tobe optimum and minimum because beyond 400 �L, volume is toohigh as substrate availability to enzyme is more results in higherundesirable background CL signals and wastage of substrate, there-fore 800 �L was ruled out. Comparatively lower CL signals wereobtained with lower volume of CDP Star due to incomplete immer-sion of dipstick in cuvette results in less substrate available forenzyme to generate CL signals as shown in Fig. 4.

3.5. Dipstick based immuno-CL assay

After the generation of antibody against vitamin B12 ELISA wasperformed for its sensitivity and specificity. The limit of detectionis 10 ng mL−1 and linear up to 100 �g mL−1 with a regression coef-ficient of 0.989 [23], but it suffer from drawbacks such as timeconsuming and cost, in order to resolve the issues, dipstick basedformat was followed for its analysis. The principle is competitivebinding between the immobilized vitamin B12 antibodies with freevitamin B12 and vitamin B12–ALP conjugated on NC membranecontaining strips was the key principle behind the detection ofvitamin B12 by immuno-CL based immunoassay. Hence when thevitamin B12 concentration is less, more conjugate bind to the stripand more CL are observed. The signal generated during reactionwas inversely proportional to the presence of vitamin B12 in thesamples.

Studies on different dilutions of vitamin B12–ALP conjugatewere done to obtain optimal CLU readings, and a 50 �g mL−1 wasfound to be optimal for vitamin B12 detection. Further optimizationof antibody concentrations showed that 100 ng �L−1 was suitablefor the detection of varying concentrations of vitamin B12 using

Fig. 4. Effect of substrate CDP star volume. The reaction mixtures contained anti-body of 100 ng �L−1 by direct spotting on to the surface of NC membrane followedby blocking with 2% skimmed milk. Strips were dipped in 50 �g mL−1 concentrationof vitamin B12–ALP conjugate to check for optimum CL generation using differentvolume from 100, 200, 400, 800 �L of 12.5 �M of concentration of CDP star in aluminometer. The photons generated as a result of the reaction were recorded for aperiod of 10 min using the luminometer and converted into a readable format usinghexa terminal software as CLU. Data were derived from triplicate assays (n = 3) witherror bars of 5%.

an optimized vitamin B12–ALP conjugate. This method was able todetect vitamin B12 at 1 ng mL−1 with range of 1 to 500 ng mL−1;below 1 ng mL−1, the signals were not reproducible and no signif-icant difference was observed in the CL response. In the dipsticktechnique, CLU response was generated by bound vitamin B12–ALPconjugate with CL reaction, and the results obtained were inverselyproportional to vitamin B12 concentration. For different concen-trations of vitamin B12, different CLU responses were obtained,as shown in the inset picture in Fig. 5. In Fig. 5, the CL response

Fig. 5. The response graph and the logarithmic plot of concentration of vitaminB12 vs CLU in dipstick based immunochemiluminescence assay. The reaction mix-tures contained 100 ng �L−1 antibodies by direct spotting on to the surface of NCmembrane followed by blocking with 2% skimmed milk. Strips were dipped in dif-ferent concentration of derivatized vitamin B12 from 1, 10, 100 and 500 ng mL−1

followed by dipping in 50 �g mL−1 concentration of vitamin B12–ALP conjugate tocheck for optimum chemiluminescence generation using 12.5 �M (400 �L) concen-tration CDP star in a luminometer. The photons generated as a result of the reactionwere recorded for a period of 10 min using the luminometer and converted intoa readable format using hexa terminal software as CLU. Data were derived fromtriplicate assays (n = 3) with error bars of 5%.

112 L.S. Selvakumar, M.S. Thakur / Analytica Chimica Acta 722 (2012) 107– 113

Table 1Precision of spiked derivatized vitamin B12 concentration estimates determinedwith a dipstick based immuno-CL. Samples were assayed in triplicate of 5 assaysper day over 5 days (n = 3).

Sample (ng mL−1) 1 10 100

Triplicates/day 5 5 5Days 5 5 5N 25 25 25Mean (ng mL−1) 0.82 9.34 99.36Recovery (%) 90 93.4 99.36%CV (intra assay) 0.1 0.03 0.003%CV (inter assay) 0.1 0.2 0.05

for each vitamin B12 concentration was plotted, and the area cov-ered under each concentration was calculated by taking the totalreading from the initial to final CLU as an integrated value that ispresented as a single line graph. In the inset picture, the y-axis isrepresented as CLU, and in the main figure, it represents integratedCLU against vitamin B12 concentration in ng mL−1. Different CLUsignals were monitored over a fixed time period (450 s) for eachvitamin B12 concentration. From the response graph (Fig. 5, insetpicture) obtained for different dilutions of vitamin B12, a linear stan-dard graph of vitamin B12 was constructed (Fig. 5), and it was foundthat a good regression value (R2 = 0.9897) was obtained in the rangeof 1–500 ng mL−1.

3.6. Comparison between colorimetric and CL detection

The optimized CL enzyme immunoassay was compared with aconventional colorimetric method. By comparing the dose responsecurves shown in Fig. 5, it can be observed that the CL methodprovided a lower detection limit with respect to the colorimetricassay (i.e. 10 ng mL−1 to 1 ng mL−1, respectively). A further advan-tage obtained by using the CL detection is the rapidity of the assay,since the CL signal can be measured immediately after substrateaddition, while the colorimetric assay requires a 20–30 min incu-bation step, as well as an enzyme activity blocking step, prior tosignal detection. Indeed, thanks to the glow type emission kinet-ics of the enhanced CL substrate, the steady state light emission isreached 2–3 min after substrate addition and it is maintained forat least 15 min, thus allowing easy handling and standardizationof the experimental conditions. In this particular experiments, ALPwas chosen as enzyme due to its stable CL response when compareto Horse Radish Peroxidase (HRP) as its counterpart. The CL gener-ated by HRP reactions with urea–hydrogenperoxide (Urea–H2O2)and luminol was not reproducible due to the unstable nature ofUrea–H2O2. But in case of ALP, substrate CDP star is very stableand gives stable CL response as shown in Fig. 5. In addition to that,the ALP tagged dipstick is easy to dip in cuvette containing singlesubstrate CDP star where as in case of HRP, two reactants are neces-sary, i.e. luminol and Urea–H2O2 which leads to non-reproducibleCL response. Therefore ALP enzyme could be an ideal tool in dipstickbased immuno-CL assay format.

3.7. Precision

In precision studies, samples were spiked with vitamin B12 andextracted using XAD-2 amberlite and derivatized at three differentconcentrations and each measured five times in triplicate on fivedifferent days are shown in Table 1. Recovery was observed to be90–99.36% with coefficients of variation between 0.003 and 0.2%for both inter and intra assay. But in case of energy drinks, vitaminB12 was to be extracted and derivatized for precision studies.

Table 2Recovery of externally added (ng mL−1) derivatized vitamin B12 in energy drinks wasanalyzed using dipstick method. Data were derived from triplicate assays (n = 3).

Energy drinks Added (ng mL−1) Found (ng mL−1) RSD (%) Recovery (%)

Sample 1 0 9.26 46.410 18.4 5.22 9250 58.6 1.52 97.6

100 109.4 0.5 99.4

Sample 2 0 8.98 26.310 18.86 8.9 9450 58.8 2.52 98

100 109.4 0.8 99.4

Table 3Analysis of vitamin B12 in energy drinks using ELISA in comparison with dipstickbased immuno-CL. The average of five measurements (±D). Amount of vitamin B12

labeled in energy drinks are 2 �g 100 mL−1.

Energy drinks Amount labeled(�g 100 mL−1)

Amount found

ELISA Dipstick Immuno-CL

Sample 1 2 1.72 (±0.42) 1.74 (±0.41)Sample 2 2 1.96 (±0.28) 1.90 (±0.41)

3.8. Accuracy

The accuracy was assessed by analyzing the energy drinks beforeand after the addition of derivatized vitamin B12. On average,99–101% of added derivatized vitamin B12 was recovered as shownin Table 2.

3.9. Comparison study

Two different brands of energy drinks labeled with vitaminB12 samples were compared using the ELISA and dipstick basedimmuno-CL. The results obtained by the ELISA and HPLC agreedwell with the labeled values for vitamin B12 in energy drinks asshown in Table 3. A comparison of two different immunologicalmethods, ELISA and dipstick based immuno-CL, demonstrates theusefulness of the vitamin B12 immunoassay as shown in Fig. 6.There was no difference between the two methods as determined.To compare the results of the two different immunological meth-ods (ELISA vs dipstick based immuno-CL) at different concentrationranges, the ELISA concentration values were well correlated tofall within the detection range of the dipstick based immuno-CLmethod.

Fig. 6. Linear regression graph of correlation between ELISA and HPLC. Data werederived from triplicate assays (n = 3) with error bars of 5%.

L.S. Selvakumar, M.S. Thakur / Analytica Chimica Acta 722 (2012) 107– 113 113

4. Conclusion

From the studies it can be proved that dipstick based detectioncan be better alternative to conventional immunological methodsuch as ELISA in terms of its cost, time duration, robustness and easyfor handling. Dipstick technique coupled with CL for the sensitivedetection will be an important aspect of the biosensor for cheaperas well as less time consuming and field applicable techniques usedfor the trace level detection of vitamin B12 in food and pharmaceu-tical industries. The methods are reliable and cost-effective, andhave several advantages over standard gas chromatography, HPLC,ELISA, and enzymatic methods. As a futuristic approach, this dip-stick technique can be further applied for nanotechnological basedanalysis of vitamin B12 by using quantum dots and gold nanoparti-cles for visual detection of analytes in food samples.

Acknowledgments

We thank the Director of the Central Food TechnologicalResearch Institute (Mysore, India) for providing constant encour-agement. Sagaya Selva Kumar L is thankful to the Council ofScientific and Industrial Research for providing a Senior ResearchFellowship.

References

[1] J.P. Sherry, Chemosphere 34 (1997) 1011–1025.[2] S.H. Paek, S.H. Lee, J.H. Cho, Y.S. Kim, Methods 22 (2000) 53–60.[3] M. Lisa, R.S. Chouhan, A.C. Vinayaka, H.K. Manonmani, M.S. Thakur, Biosens.

Bioelectron. 25 (2009) 224–227.

[4] Y.J. Cho, D.H. Lee, D.O. Kim, W.K. Min, K.T. Bong, G.G. Lee, J.H. Seo, J. Agric. FoodChem. 53 (2005) 8447–8451.

[5] Z. Schneider, A. Stroinski, Comprehensive B12, Walter de Gruyter, New York,1987.

[6] S.T. Hodson, S. Subramanian, J.R. Allen, J. Assoc. Off. Anal. Chem. 67 (1984)994–998.

[7] R.C. Oh, L.D. Brown, Am. Fam. Physician 67 (2003) 979–986.[8] A. Kumudha, L.S. SelvaKumar, M.S. Thakur, G.A. Ravishankar, R. Sarada, J. Agric.

Food Chem. 58 (2010) 9925–9930.[9] B.P. Kelleher, J.M. Scott, S.D. O’Broin, Med. Lab. Sci. 47 (1990) 90–96.

[10] P.J. Casey, K.R. Speckman, F. Ebert, W.E. Hobbs, J. Assoc. Off. Anal. Chem. 65(1982) 85–88.

[11] K. Muhammed, D. Briggs, G. Jones, Food Chem. 48 (1993) 431–434.[12] C.K. Markopoulou, K.A. Kagkadis, J.E. Koundourellis, J. Pharm. Biomed. Anal. 30

(2002) 1403–1410.[13] P. Moreno, V. Salvado, J. Chromatogr. A 870 (2000) 207–215.[14] P. Vinas, I. Campillo, N. Lopez-Garcia, M. Hernandez-Cordoba, Anal. Chim. Acta

318 (1996) 319–325.[15] M. Morita, T. Uehiro, K. Fuwa, Anal. Chem. 52 (1980) 349–351.[16] H. Karsilayan, Spectrochim. Acta 52 (1996) 1163–1168.[17] B. Morelli Freseniu, J. Anal. Chem. 354 (1996) 97–102.[18] H.B. Li, F. Chen, Y. Jiang, J. Chromatogr. A 891 (2000) 243–247.[19] S.A. Baker, Spectrochim. Acta B 55 (2000) 1823–1832.[20] L.S. SelvaKumar, R.S. Chouhan, M.S. Thakur, Anal. Biochem. 388 (2009)

312–316.[21] A. Kalman, I. Caelen, J. Svorc, J. AOAC Int. 89 (2006) 819–825.[22] E.M. Akita, S. Nakai, J. Immunol. Methods 160 (1993) 207–214.[23] L.S. Selvakumar, M.S. Thakur, Anal. Biochem. 418 (2011) 238–246.[24] R.S. Chouhan, V.S.R. Kumar, C. Raman Suri, K. Ravindranathan Thampi, M.S.

Thakur, J. AOAC Int. 93 (2010) 28–35.[25] R.S. Chouhan, K. Vivek Babu, M.A. Kumar, N.S. Neeta, M.S. Thakur, B.E. Amitha

Rani, A. Pasha, N.G. Karanth, N.G.K. Karanth, Biosens. Bioelectron. 21 (2006)1264–1271.

[26] A.L. Rose, T.D. Waite, Anal. Chem. 73 (2001) 5909–5920.[27] J. Wang, C. Zhang, H. Wang, F. Yang, X. Zhang, Talanta 54 (2001) 1185–1193.

Nano RNA aptamer wire for analysis of vitamin B12

L.S. Selvakumar, M.S. Thakur ⇑Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (A Constituent Laboratory of the Council of Scientific and Industrial Research,CSIR), Mysore 570020, India

a r t i c l e i n f o

Article history:Received 5 February 2012Received in revised form 16 May 2012Accepted 20 May 2012Available online 29 May 2012

Keywords:Biosensor20-Fluoro modified RNA aptamerGold nanoparticlesVitamin B12

a b s t r a c t

A simple and stable RNA aptamer-based colorimetric sensor for the detection of vitamin B12 using goldnanoparticles (AuNPs) has been proposed. Vitamin B12 belongs to the B vitamin group and prevents per-nicious anemia, which is caused by vitamin B12 deficiency. A highly stable RNA aptamer that binds tovitamin B12 was employed by structural modification of 20-hydroxyl group of ribose to 20-flouro in allpyrimidines indicated in lowercase in 35-mer aptamer (50 GGA Acc GGu GcG cAu AAc cAc cuc AGuGcG AGc AA 30). Aggregation of AuNPs was specifically induced by desorption of the vitamin B12 bindingRNA aptamer from the surface of AuNPs as a result of the aptamer–target interaction, leading to the colorchange from red to purple. The level of detection of vitamin B12 was 0.1 lg/ml by successful optimizationof the amount of the aptamer, AuNPs, salts, and stability of the aptamer. Analysis of vitamin B12 was car-ried out, and the observed recovery was 92 to 95.3% with a relative standard deviation in the range of2.08 to 8.27%. The results obtained were compared with those of the ultraviolet–visible (UV–vis) spec-trometry method. This colorimetric aptasensor is advantageous for on-site detection with the naked eye.

� 2012 Elsevier Inc. All rights reserved.

Aptamers (DNA or RNA) have attracted an increasing amount ofinterest in the development of sensors for drugs, proteins, aminoacids, organic and inorganic molecules, and even supramolecularcomplexes such as viruses and cells [1,2]. Aptamers have potentialapplication as a recognition element in analytical and diagnosticassays because aptamers can be easily screened, designed, andevolved by an in vitro selection process known as systematic evo-lution of ligands by exponential enrichment (SELEX)1 [3,4]. Relativeto antibodies, aptamers have several advantageous properties,including stability, low cost, and ease of synthesis [5].

Natural RNA or DNA is nuclease sensitive; therefore, nuclease-resistant oligonucleotides are generated for wide applications inbiosensors. To generate nuclease-resistant oligonucleotide li-braries, we have used modified RNA in which the 20-hydroxy(-OH) group in the pyrimidine nucleotides was substituted withthe fluoro (F–) functionality. This modification is known to imparta substantial degree of mechanism-based protection against themajority of endo- and ribonucleases [6]. Importantly, the 20-fluorosubstitution is compatible with the enzymatic steps of SELEX.Thus, SELEX experiments with libraries carrying such modifica-tions can lead to nuclease-resistant ligands. 20-Fluoro-modified

RNA aptamers potentially have more rigid structures than 20-amino aptamers because of their stronger intramolecular helices,leading to thermodynamically stable secondary structure andhigher affinities.

We have chosen to focus on the vitamin B12-specific aptamerisolated by Lorsch and Szostak [7] as a model system for biosensordevelopment. Adenosylcobalamin (the biologically active form ofvitamin B12) is believed to be one of the most evolutionarily an-cient enzyme cofactors and has been postulated to play a key rolein the transition from RNA-based biology to modern DNA and pro-tein-dominated biology [8]. Using in vitro selection from a pool of1015 random sequence molecules, Lorsch and Szostak were able toisolate one RNA molecule with a 35-nt pseudo-knot that specifi-cally recognized vitamin B12. The aptamer bound cyanocobalaminwith relatively high affinity (Kd � 90 nM), suggesting that a largenumber of specific interactions stabilized the RNA–ligand complex.The aptamer requires 1 M lithium chloride for proper folding andbinding because lithium ions form tight complexes with phos-phates, leading to more covalent binding instead of ionic pairing[9]. Further lithium ions help in the neutralization of charge back-bone of RNA, enabling RNA to fold into a more compact and stabi-lized structure. But in our case, we might not be able to use lithiumions because doing so leads to aggregation of gold nanoparticles(AuNPs) as such. So, experiments were carried out in the absenceof lithium ions and without compromising on their sensitivity.

In addition to its evolutionary importance, vitamin B12 is animportant biomolecule present in foods and medicines. Naturaland commercially known cyanocobalamin is a stable form of

0003-2697/$ - see front matter � 2012 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ab.2012.05.020

⇑ Corresponding author. Fax: +91 821 2517233.E-mail addresses: msthakur@cftri.res.in, msthakur@yahoo.com (M.S. Thakur).

1 Abbreviations used: SELEX, systematic evolution of ligands by exponentialenrichment; AuNP, gold nanoparticle; HPLC, high-performance liquid chromatogra-phy; DEPC, diethylpyrocarbonate; SPR, surface plasmon resonance; UV–vis, ultravi-olet–visible; DDW, double distilled water; ssDNA, single-stranded DNA.

Analytical Biochemistry 427 (2012) 151–157

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Analytical Biochemistry

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vitamin B12. Vitamin B12 is an essential nutrient for the mainte-nance of myelin sheath surrounding nerve cells in humans [10].It also promotes growth, cell development, and fat and carbohy-drate metabolism, and it is essential for the rapid synthesis ofDNA during cell division, particularly the bone marrow tissueresponsible for red blood cell formation [11]. Animals and plantsare unable to synthesize vitamin B12, and it is synthesized solelyby microorganisms. It is found mainly in animal sources such asmilk, meat, eggs, fish, and oysters. The daily requirement of vita-min B12 is very low when compared with those of other vitamins[12]. Deficiency of vitamin B12 leads to megaloblastic anemia, neu-rological disorders, and pernicious anemia. Hence, accurate, spe-cific, efficient, and rapid estimation of vitamin B12 at a sensitivelevel is highly desirable [13]. There are various techniques thathave been used extensively to detect vitamin B12 such as microbi-ological assays that use Lactobacillus leichmannii as a test organism[14], high-performance liquid chromatography (HPLC) [15], fluo-rimetry assay [16], capillary electrophoresis [17], enzyme-linkedimmunosorbent assay (ELISA) [18], and chemiluminescence assay[19]. These conventional methods are time-consuming, nonspe-cific, less sensitive, and expensive. To overcome these issues, apta-mers are offered as ideal candidates for use in biosensorapplications. The potential use of aptamers in biosensor applica-tions is further enhanced on their conjugation to nanoparticles.

Colorimetric aptasensors using AuNPs have been considered asan on-site detection method with high specificity and sensitivitybecause of their easy preparation, simple operation, and detectionof colorimetric signal with the naked eye [20]. In previous research,AuNPs were used extensively as important colorimetric materialsbecause they have strong distance-dependent optical propertiesthat are used for aptamer-based biosensors. Zhao et al. [21] andWang et al. [22] detected adenosine and potassium ions based sep-arately on a non-crosslinking mechanism of AuNP aggregation, andLu’s group [23–25] worked on detection of adenosine and cocainebased on a crosslinking mechanism of AuNPs by hybridizing apta-mers with complementary sequences. Similarly, Pavlov et al. [26]used aptamer-functionalized AuNPs as a catalytic label for ampli-fied detection of thrombin in solution and on surface. Recently,Song and co-workers focused on colorimetric detection of kanamy-cin using AuNPs and aptamers [27]. There are several reports ofsuccessful DNA aptamer-based sensors using AuNPs. In contrast,only one RNA aptamer-based sensor for theophylline has beendeveloped and suffers from low stability [28], and our currentstudy it has been further improved with 20-fluoro modificationsof RNA aptamer for vitamin B12 detection. RNA aptamer for vitaminB12 is known and, hence, provided a known starting point for ourstudies. In general, RNA forms diverse three-dimensional structureformation with high affinity and specificity and makes the screen-ing procedure easier for isolation of RNA aptamers from the RNAlibrary [29]. In the proposed colorimetric aptasensor, there is a tar-get-specific aggregation of AuNPs due to interaction of aptamers toanalyte after physical adsorption of aptamers on AuNPs [30]. Wesuccessfully conducted the target vitamin B12-specific aggregationof AuNPs as a result of aptamer–vitamin B12 interaction.

Materials and methods

Reagents

Vitamin B12 RNA aptamer sequence (50 GGA ACC GGU GCG CAUAAC CAC CUC AGU GCG AGC AA 30) was adapted from a previousreport [7]. Furthermore, all pyrimidines are 20-flouro modifiedand were obtained from TriLink Biotechnologies (San Diego, CA,USA). Vitamin B12, diethylpyrocarbonate (DEPC), gold(III) chloride,trisodium citrate, and silver nitrate were procured from Sigma–

Aldrich (St. Louis, MO, USA). All stock solutions were prepared inDEPC-treated water and further diluted accordingly.

Apparatus and principle of detection of vitamin B12 using proposedmethod

Spectral analysis of AuNPs was performed in the range from 300to 700 nm using a Shimadzu UV-1601 spectrophotometer (Kyoto,Japan). Fluorimetric analysis of aptamer was performed in theemission range from 250 to 500 nm at an excitation of 260 nmusing a Shimadzu RF-5301 PC spectrofluorometer.

We demonstrate that change in color of AuNPs can be used as asimple, easy-to-use assay probe that does not need precise timingand is visible to the naked eye without the need for sophisticatedinstruments to detect vitamin B12. Solutions of AuNPs are red incolor due to their specific and size-dependent surface plasmon res-onance (SPR) absorption at 520 nm. The addition of salt createdelectrostatic repulsion between negatively charged AuNPs and re-sulted in aggregation of AuNPs that led to a red-to-purple colorchange, and an additional absorption peak was observed above600 nm. We then treated AuNPs with the vitamin B12 RNA aptamerfor a few minutes both in the presence and in the absence of vita-min B12. On the addition of salt, the former solution showed a colorchange from red to purple due to the presence of vitamin B12,whereas the latter solution retained its original red color due to ab-sence of vitamin B12, as shown in Scheme 1. Aggregation of AuNPswas specifically induced by desorption of the vitamin B12 bindingRNA aptamer from the surface of AuNPs as a result of the apt-amer–target interaction, leading to the color change from red topurple.

Solution preparation

A stock solution of vitamin B12 was prepared by dissolving 1 mgof pure crystalline B12 in 1 ml of B12 binding buffer (Na–Hepes.pH 7.4). A stock solution of 1 mM RNA aptamer was prepared bydissolving 11 mg of desalted RNA aptamer in 1 ml of DEPC-treatedwater. Further dilutions of this stock solution were prepared inHepes buffer to obtain 5- to 100-lM aptamer concentrations. Anaqueous solution of monodisperse quasi-spherical AuNPs wasprepared by a modified Turkevitch and co-workers method[31,32]. A total of 45 ml of Milli-Q water was taken in a reactionflask and refluxed for 10 min with 5 ml of 0.1% tetrachloroauricacid, 2 ml of 1% trisodium citrate, and 42.5 ll of 0.1% silver nitrate.Tetrachloroauric acid, trisodium citrate, and silver nitrate weremixed together in a separate beaker and incubated for 5 min andadded dropwise into the reaction flask. The reduction of gold metalions (Au3+) to yield AuNPs (Auo) was confirmed by the appearanceof a dark cherry red color. Colloidal AuNPs were stored at 4 �C.The size and concentration of AuNPs were determined using anultraviolet–visible (UV–vis) spectrophotometer [33]. A stock solu-tion of 1 M NaCl was prepared by dissolving 40 mg in 1 ml ofDEPC-treated water and, accordingly, was diluted for optimizationstudies. The effect of incubation on binding and stability waschecked using the fluorimeter during the analysis. All samplesand standard solutions were prepared and stored in a brown cali-brated flask at 4 �C.

Analysis of vitamin B12 in pharmaceutical samples

A mixture of total volume (300 ll) consisting of an optimizedconcentration of AuNPs and aptamer was shaken mildly for10 min at room temperature and incubated for 10 min by adding100 ll of different concentrations of vitamin B12 (0.1, 0.5, 1, 5,and 10 lg/ml). After the slow addition of optimized concentrationof NaCl into the incubated sample, color and spectra changes were

152 Aptamer wire for analysis of vitamin B12 / L.S. Selvakumar, M.S. Thakur / Anal. Biochem. 427 (2012) 151–157

observed by the UV–vis spectrophotometer. Vitamin B12 injectionampoules having a concentration of 0.334 mg/ml were diluted to100 ml with water, making a stock solution of 10 lg/ml. At least6 numbers of each of the capsules and tablets containing 15 lgof vitamin B12 were taken (equivalent to 90 lg of vitamin B12)and diluted to 9 ml, giving a stock of 10 lg/ml, which was sus-pended in double distilled water (DDW) and used directly for theproposed and spectrophotometric method. For reference, pharma-ceutical preparations were made to approximate 10-lg/ml solu-tions, which were then determined at 273, 361, and 551 nmusing UV–vis spectroscopy.

Results and discussion

Preparation and spectrophotometric characterization of AuNPs

PreparationDuring the preparation of colloidal gold, tetrachloroauric acid

was reduced due to the transfer of electrons from the carboxylgroup of trisodium citrate and Au3+ ions, leading to the formationof Auo. This metallic gold then nucleates and grows to form AuNPsand is subsequently capped and stabilized by the citrate ions. Theconcentration of trisodium citrate used in its synthesis dictates thesize of AuNPs and reduces gold chloride to AuNPs. The color of thesolution changes from pale yellow to a cherry red color. A minuteconcentration of silver nitrate was added to get uniform nucleationduring the preparation of AuNPs [32].

SpectrophotometryThe size and concentration of AuNPs were determined from the

UV–vis spectrum of AuNPs [33]. From the spectrophotometric data,the following values were obtained:

kSPR ¼ 504 nm

A450 ¼ 0:914

ASPR ¼ 1:495

Diameter of AuNPs ¼ ASPR=A450:

The ratio of absorbance of AuNPs at the SPR peak (ASPR) to theabsorbance at 450 nm (A450) was calculated and found to be1.6356, which corresponds to an AuNP size (diameter) of 14 to16 nm, so an average size of 15 nm is taken for further studies.The concentration of the AuNPs was calculated using the formula:

C ¼ A450=e450;

where e450 is the molar decadic extinction coefficient at 450 nm.The average value of e450 was found to be 2.203 � 108 M–1 cm–1.By substituting the value in the above equation, we get the concen-tration of the gold sol as 4.19 � 10–9 M.

Effect of aptamer molar ratio on AuNPs

It has already been reported that uncoiled single-stranded DNA(ssDNA) can be adsorbed on AuNPs due their bases facing the AuN-Ps, leading to electrostatic interaction between the bases of ssDNAand AuNPs. AuNPs in the absence of targets are normally stabledue to the adsorbed ssDNA aptamers even if salt is added [30].But, as expected, the targets specific to the aptamers induced theadsorbed aptamers to detach from AuNPs and resulted in the sub-sequent aggregation of AuNPs, leading to the color change from redto purple. In this study, the 35-mer size of vitamin B12 binding 20-flouro-modified RNA aptamer was used for the stabilization ofAuNPs. The stability of AuNPs can be maintained by the aptamersadsorbed on them, preventing the salt-induced aggregation. Assaywas done to optimize the minimum amount of aptamer needed tostabilize AuNPs, as shown in Fig. 1A. The resultant graph showsthat most of the AuNPs get aggregated at an aptamer concentrationless than 25 lM. This indicates that lower concentrations ofaptamers are not sufficient to shield the AuNPs, leaving more freeAuNPs for salt-induced aggregation, whereas AuNPs do not getaggregated at aptamer concentrations higher than 25 lM, indicat-ing that aptamers are completely shielding the AuNPs. Thus, a25-lM aptamer concentration was selected as optimum becausevisually and spectrophotometrically distinct color change wasobserved. The color change of the AuNPs is due mainly to varyinginterparticle plasmon coupling during aggregation, resulting inplasmon band shift.

Scheme 1. Vitamin B12 RNA aptamer and colorimetric detection of vitamin B12-induced structural variation. (Adapted and modified from Wang et al. [22]).

Aptamer wire for analysis of vitamin B12 / L.S. Selvakumar, M.S. Thakur / Anal. Biochem. 427 (2012) 151–157 153

Effect of AuNPs on RNA aptamer concentration

To increase the sensitivity of vitamin B12, an assay was per-formed to optimize the minimum amount of AuNPs needed to ad-sorb on 25-lM concentration of aptamer with observable spectraand colorimetric changes. As shown in Fig. 1B, most of the AuNPs

get aggregated at amounts higher than 300 ll of 4.19 � 10–9 Mconcentration; this indicates that the aptamer concentration isnot sufficient to shield the complete AuNPs, leaving more freeAuNPs that lead to salt-induced aggregation, whereas at loweramounts of AuNPs the aptamer completely shields the AuNPsand does not allow salt-induced aggregation. Thus, 300 ll of AuNPs

Fig.1. (A) Absorbance spectra for an optimization of aptamer concentration (10, 25, and 50 lM) to AuNPs in a wavelength range from 400 to 700 nm. (B) Absorbance spectrafor an optimization of AuNP concentration (200, 300, 400, and 500 ll) to aptamer in a wavelength range from 400 to 700 nm. (C) Absorbance spectra for an optimization ofsalt concentration (0.1, 0.25, 0.5, and 1 M) in a wavelength range from 400 to 700 nm. (D) UV–vis absorbance spectra of 25 lM aptamer, 0.1 lg/ml vitamin B12, and 300 ll ofAuNPs after different incubation time periods (0, 15, 30, and 60 min). (E) Fluorimetric detection of aptamer stability after particular time intervals (0, 3, 6, 9, 12, and 24 h). (F)Fluorimetric emission spectra of aptamer with different concentrations of vitamin B12 (0.1, 1, and 10 lg/ml).

154 Aptamer wire for analysis of vitamin B12 / L.S. Selvakumar, M.S. Thakur / Anal. Biochem. 427 (2012) 151–157

was selected as optimum because visually and graphically distinctcolor change was observed.

Effect of salt concentration

An assay was performed to optimize the optimal concentrationof salt required for the aggregation of AuNPs and desired colori-metric changes observed as salt increases the ionic strength anddecreases the interparticle distance among the AuNPs, leading tochange in color from red to purple due to aggregation. From the re-sults of spectra observed at different concentrations of NaCl, asshown in Fig. 1C, it was found that less than 0.5-M NaCl concentra-tions of AuNPs are not aggregated effectively, whereas at NaCl con-centrations higher than 0.5 M effective aggregation of AuNPs wasobserved. Thus, among all of the concentrations of salt, 0.5 M NaClwas selected as optimum because visually and spectrophotometri-cally distinct color change was observed. The salt-induced aggrega-tion of AuNPs is due mainly to Van der Waals forces of attractionagainst repulsion between the AuNPs. Therefore, the addition ofa sufficient amount of salt screens the repulsion between the neg-atively charged AuNPs and leads to the aggregation responsible forthe color change.

Incubation studies

Incubation studies were done to optimize the time required tobind the aptamer to vitamin B12 from AuNPs. From the results ofspectra observed at different incubation times, as shown inFig. 1D, it was found that at 0 min there was no difference in spec-tra after the addition of salt immediately because sufficient reac-tion time was not given to the aptamers to dissociate fromAuNPs and bind to vitamin B12. Therefore, visually the color ob-served was red. After 15 min of incubation, the aptamers boundto AuNPs get dissociated and bind with vitamin B12 because apta-mers are highly specific for the target, leaving AuNPs for salt-induced aggregation, and the color changed to purple. Thus, amongall of the incubation studies, 15 min was selected as the optimumtime.

Stability of RNA aptamer at different incubation times usingfluorimeter

Incubation studies were done to determine the stability of RNAaptamer after incubating at different time periods at room temper-ature, as shown in Fig. 1E. The results obtained show that at 0 haptamer showed optimal fluorescent intensity. As time increased,there was a decrease in fluorescent signals because the aptamerswere exposed to nuclease attack due to working in an environmentwithout any major RNA safety precautionary measures. Therefore,there may be a degradation of aptamers, resulting in less emissionintensity. It is a well-known phenomenon that native RNA is notstable for even seconds, but 20-flouro-modified RNA is stable formore than 15 h [34,35]. In our case, the overall time of analysisis only 15 min, for which 20-fluoro-modified aptamers are betterthan native RNA aptamers without losing their activity. The sameaptamer would be carried for biosensor application that requiresstringent and different environmental conditions; therefore, 20-flu-oro-modified RNA aptamers may be well suited for such analyticalapplications.

Binding studies of RNA aptamer to vitamin B12 using fluorimeter

Similar binding was confirmed using the emission intensity ofRNA aptamer with different concentrations of vitamin B12, asshown in Fig. 1F. The resultant graph shows that the control, con-taining only aptamer, had higher emission intensity as compared

with aptamer with different vitamin B12 concentrations. At higherconcentrations of vitamin B12 there is less emission intensity, andat lower concentrations higher emission intensity was observed.This shows that emission intensity is indirectly proportional tovitamin B12 concentration. The emission of aptamer is due mainlyto its bases. When aptamer and vitamin B12 get bound, their emis-sion gets decreased due to conformational changes in the aptamerand reduced exposure of bases on its surface. It is a well-knownphenomenon that native RNA or DNA has less emission propertiesdue to weak fluorophore nitrogen bases [36], but in our case weobserved distinguishable emission intensities due to incorporationof 20-fluoro moiety in all pyrimidines. It is proved that aromaticsubstitution with fluorine generally leads to increased fluorescentproperties, due partly to the greater absorption of these com-pounds in the UV region as compared with the unsubstituted mol-ecules [37].

Performance of proposed method for vitamin B12 determination inpharmaceutical preparations

Based on the understanding on the interaction between apt-amer and AuNPs, the colorimetric assays appearing as the resultsof AuNP aggregation at the different concentrations of vitaminB12 are shown in Fig. 2. The resultant graph shows that vitaminB12 at the highest concentration of 10 lg/ml leads to more bindingof aptamer with it because aptamers are highly specific and havehigh affinity toward the target, leaving AuNPs for salt-inducedaggregation, and color turned to purple. In the case of 1 lg/ml vita-min B12 concentration, few aptamers get bound with vitamin B12

and remain with AuNPs, leaving free AuNPs to get aggregated par-tially, and so color turned to a lighter purple. The control samplewith no vitamin B12 retains red color due to physical adsorptionof aptamer to AuNPs, and so a sharp absorbance peak was obtainedat a wavelength of 520 nm. UV–vis studies provided quantitativeresults clearly showing that adsorption at 520 nm gradually de-creased, whereas adsorption at 640 nm increased. This blue shiftin the SPR absorption suggested the formation of large-sized aggre-gates of AuNPs. Significantly, color change is visible at as low as0.1 lg/ml vitamin B12, suggesting that AuNPs are sensitive probesfor aptamer structures. As can be seen in Fig. 2, the spectral and

Fig.2. UV–vis absorption spectra for limit of detection of different concentrations ofvitamin B12 (0.1, 0.5, 1, 5, and 10 lg/ml) using RNA aptamer and AuNPs. Inset: Plotof absorption ratio (A520/A640) versus vitamin B12 concentration. Colored photo-graphs of 300 ll of 15-nm AuNPs stabilized by the aptamer (100 ll, 25 lM) afterthe addition of salt (50 ll, 0.5 M NaCl) in the presence of different concentrations ofvitamin B12 are shown (from left to right). (For interpretation of the reference tocolor in this figure legend, the reader is referred to the Web version of this article.).

Aptamer wire for analysis of vitamin B12 / L.S. Selvakumar, M.S. Thakur / Anal. Biochem. 427 (2012) 151–157 155

color changes of AuNP solution were measured up to as low as0.1 lg/ml vitamin B12, depending on the measurement method,either spectrophotometry or the naked eye. For convenient analy-sis of the spectral and color changes, the absorbance ratios at A520/A640 were plotted and found to be well matched with the spectraand visual observation. The data points in the Fig. 2 inset showthe A520/A640 ratio versus the vitamin B12 concentration (from 10to 0.1 lg/ml) of the solution consisting of 25 lM vitamin B12

RNA aptamer, 300 ll of 4.19 � 10–9 M AuNP concentration, and0.5 M NaCl. The higher the vitamin B12 concentration, the greaterthe change in color and the higher the A520/A640 ratio. In this exper-iment, vitamin B12 was easily detected at 0.1 lg/ml, but it was notpossible to detect it below this value. In addition, the degree ofAuNP aggregation did not change in the presence of more than10 lg/ml vitamin B12. Furthermore, the well-fitted curve fromA520/A640 ratio data shows that the designed method is a reliablesystem for vitamin B12 detection with 10 lg/ml as the upper limitand 0.1 lg/ml as the lower limit.

The method was applied to the analysis of three preparations ofvitamin B12 (injections, tablets, and capsules) that were purchasedfrom the local market. To check the accuracy of the proposedmethod, the UV–vis spectroscopy method was used to measurethe content of vitamin B12 in all samples in triplicate. The resultsare given in Table 1. The two methods show good agreement. Tocheck the accuracy of the results, a recovery study was carriedout by comparing the concentrations found in three pharmaceuti-cal samples spiked with known amounts of vitamin B12. The overallrecovery was 92 to 95.3% with a relative standard deviation in therange of 2.08 to 8.27%, as shown in Table 2. The results obtained bythe current method agreed well with the labeled values for vitaminB12 injection, tablets, and capsules. To assess the possible analyticalapplications of the proposed method, the effects of various water-soluble vitamins and metal ion contaminants were tested by ana-lyzing a standard solution of vitamin B12 (1 lg/ml) to whichincreasing amounts of interfering species were added. A standardsolution of 1 lg/ml vitamin B12 was used to analyze nonspecificaggregation. A0 and A are the absorbance ratio (A520/A640) of theproposed method in the absence and presence of interfering spe-cies, respectively, as shown in Table 3. Tolerable concentrations,defined as the concentrations of foreign species causing less than±3% relative error DA/A (%), were examined. The results showedthat the tolerable concentration ratios of coexisting substances to1.0 lg/ml vitamin B12 were more than 1000 for Al3+, Zn2+, Pb2+,

Hg2+, Ca2+, Ag+; Mn2+, Fe2+, Mg2+, Cd2+,Co2+, Cu2+, Fe3+, Li2+, andvitamin B12 analogs and more than 3000 for water-soluble vita-mins. It can be seen that some common excipients and their poten-tial impurities in pharmaceutical preparations have no effect onthe determination of low concentrations of vitamin B12. However,metal ions are severe interferers for the determination. Because al-most no commercial pharmaceutical injections, capsules, and tab-lets contain the above interference level, the method can beapplied for the direct determination of vitamin B12 in pharmaceu-ticals. Thus, the proposed method showed good selectivity andindicates that, prior to evaluation, samples with excess metal ionsshould be taken care of by chelating with ethylenediaminetetra-acetic acid (EDTA) [38,39].

In conclusion, vitamin B12 was analyzed up to a minimum con-centration of 0.1 lg/ml using 25 lM RNA aptamer, 300 ll of4.19 � 10–9 M AuNPs, and 0.5 M NaCl concentration. ModifiedRNA aptamer was stable for up to 2 h, a feature not explored pre-viously. Compared with conventional analytical techniques such asHPLC, atomic absorption spectrometry, inductively coupled plasmamass spectrometry, radioisotope assay, microbiological assay, andfluorimetric detection, the current proposed method was sensitive,

Table 1Analysis of vitamin B12 in pharmaceutical samples using proposed method in comparison with UV–vis spectroscopy.

Pharmaceutical sample Amount labeled Amount found

Proposed method UV–vis spectrophotometric method

Multivitamin injection 0.334 0.32 ± 0.04 0.33 ± 0.06Multivitamin tablet 15 14.06 ± 0.35 14.24 ± 0.46Multivitamin capsule 15 14.44 ± 0.50 14.60 ± 0.35

Note: The averages of five measurements ± standard deviations are shown. The amount of vitamin B12 labeled in a multivitamin injection is 0.334 mg/ml, in a multivitamintablet is 15 lg/tablet, and in a multivitamin capsule is 15 lg/capsule.

Table 2Recovery of externally added (1 lg/ml) vitamin B12 in pharmaceutical samples analyzed using proposed method.

Pharmaceutical sample Added (lg/ml) Found (lg/ml) Relative standard deviation (%) Recovery (%)

Injections 0 0.926 5.591 1.840 5.22 92.0

Tablets 0 0.898 8.271 1.920 3.08 94.3

Capsules 0 0.920 4.861 1.906 2.08 95.3

Note: Data were derived from triplicate assays (n = 3).

Table 3Tolerable concentrations of coexisting substances on the absorbance ratio (A520/A640)of proposed method with respect to 1 lg/ml vitamin B12.

Interfering substance Tolerable concentration(lg/ml)

Relative error DA/A (%)

Al3+ 1300 0.60Zn2+ 1500 �0.30Pb2+ 1100 0.73Hg2+ 1300 0.50Ca2+ 1100 �0.30Ag+ 1250 �1.70Mn2+ 1150 �3.00Fe2+ 1000 0.50Mg2+ 1450 �0.28Cd2+ 1300 �1.30Co2+ 1150 0.56Cu2+ 1400 �1.23Fe3+ 1500 �1.00Li2+ 1000 0.03Vitamin B12 analogs 1000 �3.40Water-soluble vitamins 3000 �4.80

Note: Relative error DA = A0 � A, where A0 and A are the absorbance ratios in theabsence and presence of interfering species, respectively.

156 Aptamer wire for analysis of vitamin B12 / L.S. Selvakumar, M.S. Thakur / Anal. Biochem. 427 (2012) 151–157

rapid, and economical, required no expensive instrumentation, andcan be used for on-site analysis. The level of detection obtained forvitamin B12, which was 0.1 lg/ml in terms of spectral change orcolorimetric change, is still higher than the recommended dietaryallowance (RDA) value regulation (2–3 lg/100 g in food). There-fore, this optimization was conducted to achieve a lower level ofdetection than the regulation. Although this work is in its prelimin-ary stage, it could be further improved by introducing severalexisting technologies that might eventually lead to a cost-effectivebiosensor platform. Because the detection is homogeneous, it iseasily adaptable for high-throughput assays in microwell-basedplates and even automated analysis. More important, this strategyis not limited to detection of vitamin B12. In fact, RNA aptamers canbe stabilized by 20-fluoro modifications for general biosensordevelopment. Therefore, the principle described in this work canbe conveniently generalized to other aptamer systems. Eventually,we expect that it will be possible to visually detect a large numberof important analytes with unmodified AuNPs and aptamers gener-ated from SELEX.

Acknowledgments

The authors thank the director of the Central Food Technologi-cal Research Institute (Mysore, India) for providing constantencouragement. L.S. Selva kumar is grateful to the Council of Scien-tific and Industrial Research for providing a senior researchfellowship.

References

[1] E. Luzi, M. Minunni, S. Tombelli, M. Mascini, New trends in affinity sensing:aptamers for ligand binding, Trends Anal. Chem. 22 (2003) 810–818.

[2] K.M. You, S.H. Lee, A. Im, S.B. Lee, Aptamers as functional nucleic acids: in vitroselection and biotechnological applications, Biotechnol. Bioprocess. Eng. 8(2003) 64–75.

[3] A.D. Ellington, J.W. Szostak, Selection in vitro of single-stranded DNAmolecules that fold into specific ligand-binding structures, Nature 355(1992) 850–852.

[4] T. Herman, D. Patel, Adaptive recognition by nucleic acid aptamers, Science287 (2000) 820–825.

[5] D.J. Sumedha, Aptamers: an emerging class of molecules that rival antibodiesin diagnostics, Clin. Chem. 45 (1999) 1628–1650.

[6] W. Pieken, D.B. Olsen, F. Benseler, H. Aurup, H.F. Eckstein, Kineticcharacterization of ribonuclease-resistant 20-modified hammerheadribozymes, Science 253 (1991) 314–317.

[7] J.R. Lorsch, J.W. Szostak, In vitro selection of RNA aptamers specific forcyanocobalamin, Biochemistry 33 (1994) 973–982.

[8] S.A. Benner, A.D. Ellington, A. Tauer, Modern metabolism as a palimpsest of theRNA world, Proc. Natl. Acad. Sci. USA 86 (1989) 7054–7058.

[9] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, John Wiley, NewYork, 1988.

[10] V. Herbert, Vitamin B12, in: E.E. Ziegler, L.J. Filer (Eds.), Present Knowledge inNutrition, 17th ed., International Life Sciences Institute Press, Washington, DC,1996, pp. 191–205.

[11] S.P. Stabler, B12 and nutrition, in: R. Banerjee (Ed.), Chemistry andBiochemistry of B12, John Wiley, New York, 1999, pp. 343–365.

[12] S. Subramanium, J.R. Allen, Determination of pentatonic acid, biotin, andvitamin B12 in nutritional products, Anal. Chem. 67 (1984) 994–998.

[13] L.D. Brown, Vitamin B12 deficiency, Am. Fam. Physician 67 (2003) 979–986.[14] B.P. Kelleher, J.M. Scott, Cryopreservation of Lactobacillus leichmanni for

vitamin B12 microbiological assay, Med. Lab. Sci. 47 (1990) 90–95.

[15] O. Heudi, T. Kilinc, P. Fontannaz, E. Marley, Determination of vitamin B12 infood products and in premixes by reversed high performance liquidchromatography and immune affinity extraction, J. Chromatogr. A 1101(2006) 63–68.

[16] H.B. Li, F. Chen, Determination of vitamin B12 in pharmaceutical preparation bya highly sensitive fluorimetric method, Fresenius J. Anal. Chem. 368 (2000)836–838.

[17] S.A. Baker, N.J. Miller, Determination of cobalamins using capillaryelectrophoresis inductively coupled plasma mass spectrometry, Spectrochim.Acta 55 (2000) 1823–1832.

[18] L.S. Selvakumar, M.S. Thakur, Competitive immunoassay for analysis ofvitamin B12, Anal. Biochem. 418 (2011) 238–246.

[19] L.S. Selvakumar, R.S. Chouhan, M.S. Thakur, Enhancement ofchemiluminescence for vitamin B12 analysis, Anal. Biochem. 388 (2009)312–316.

[20] M. Cho, M.S. Han, C. Ban, Detection of mismatched DNAs via the bindingaffinity of MutS using a gold nanoparticle-based competitive colorimetricmethod, Chem. Commun. 38 (2008) 4573–4575.

[21] W. Zhao, W. Chiuman, M.A. Brook, Y. Li, Simple and rapid colorimetricbiosensors based on DNA aptamer and noncrosslinking gold nanoparticleaggregation, Chem. Biochem. 8 (2007) 727–731.

[22] L. Wang, X. Liu, X. Hu, S. Song, C. Fan, Unmodified gold nanoparticles as acolorimetric probe for potassium DNA aptamers, Chem. Commun. 36 (2006)3780–3782.

[23] J. Liu, Y. Lu, Adenosine-dependent assembly of aptazyme-functionalized goldnanoparticles and its application as a colorimetric biosensor, Anal. Chem. 76(2004) 1627–1632.

[24] J. Liu, Y. Lu, Fast colorimetric sensing of adenosine and cocaine based on ageneral sensor design involving aptamers and nanoparticles, Angew. Chem.Int. Ed. 45 (2006) 90–94.

[25] J. Liu, D. Mazumdar, Y. Lu, A simple and sensitive ‘‘dipstick’’ test in serumbased on lateral flow separation of aptamer-linked nanostructures, Angew.Chem. Int. Ed. 45 (2006) 7955–7959.

[26] V. Pavlov, Y. Xiao, B. Shlyahovsky, I. Willner, Aptamer-functionalized Aunanoparticles for the amplified optical detection of thrombin, J. Am. Chem. Soc.126 (2004) 11768–11769.

[27] K.M. Song, M. Cho, H. Jo, K. Min, S.H. Jeon, T. Kim, M.S. Han, J.K. Ku, C. Ban, Goldnanoparticle-based colorimetric detection of kanamycin using a DNA aptamer,Anal. Biochem. 415 (2011) 175–181.

[28] E.E. Ferapontova, M.E. Olsen, V.K. Gothelf, An RNA aptamer-basedelectrochemical biosensor for detection of theophylline in serum, J. Am.Chem. Soc. 130 (2008) 4256–4258.

[29] S.C.B. Gopinath, Methods developed for SELEX, Anal. Bioanal. Chem. 287(2007) 171–182.

[30] H. Li, L. Rothberg, Colorimetric detection of DNA sequences based onelectrostatic interactions with unmodified gold nanoparticles, Proc. Natl.Acad. Sci. USA 101 (2004) 14036–14039.

[31] J. Turkevitch, P.C. Stevenson, J. Hillier, Nucleation and growth process in thesynthesis of colloidal gold, Faraday Soc. 11 (1951) 55–75.

[32] H. Xia, S. Bai, J. Artman, D. Wang, Synthesis of monodisperse quasi-sphericalgold nanoparticles in water via silver(I)-assisted citrate reduction, Langmuir26 (2010) 3585–3589.

[33] W. Haiss, N.T.K. Thanh, J. Aveyard, D.G. Fernig, Determination of size andconcentration of gold nanoparticles from UV–vis spectra, Anal. Chem. 79(2007) 4215–4221.

[34] C.P. Nikos, B. Carol, C. Ying-Fon, J. Susan, F. Tim, J. Derek, D. Chinh, Potent 20-amino- and 20-fluoro-20-deoxyribonucleotide RNA inhibitors of keratinocytegrowth factor, Nat. Biotechnol. 15 (1997) 68–73.

[35] L. Seong-Wook, A.B. Sullenger, Isolation of a nuclease-resistant decoy RNA thatcan protect human acetylcholine receptors from myasthenic antibodies, Nat.Biotechnol. 15 (1997) 41–45.

[36] S. Udenfriend, P. Zaltzman, Fluorescence characteristics of purines,pyrimidines, and their derivatives: measurement of guanine in nucleic acidhydrolyzates, Anal. Biochem. 3 (1962) 49–59.

[37] G. Gordon, A.C. Michael, Fluorescence of the fluorinated phenylalanine, Anal.Biochem. 4 (1962) 330–336.

[38] K. Youngjin, C.J. Robert, T.H. Joseph, Gold nanoparticle based sensing of‘‘spectroscopically silent’’ heavy metal ions, Nano Lett. 1 (2001) 165–167.

[39] N. Ding, H. Zhao, W. Peng, Y. He, Y. Zhou, L. Yuan, Y. Zhang, A simplecolorimetric sensor based on anti-aggregation of gold nanoparticles for Hg2+

detection, Colloids Surf. A 395 (2012) 161–167.

Aptamer wire for analysis of vitamin B12 / L.S. Selvakumar, M.S. Thakur / Anal. Biochem. 427 (2012) 151–157 157