Advances in nano-scaled biosensors for biomedicalapplications

Jianling Wang, Guihua Chen, Hui Jiang, Zhiyong Li* and Xuemei Wang*

Recently, a growing amount of attention has been focused on the utility of biosensors for biomedical

applications. Combined with nanomaterials and nanostructures, nano-scaled biosensors are installed for

biomedical applications, such as pathogenic bacteria monitoring, virus recognition, disease biomarker

detection, among others. These na

are ideally suited to biomedical

allowing biomedical analysis with

discusses the literature published in

n

t

extremely important and has also remained a challenge.Biosensors with biological recoorganisms or biological materiadetection and monitoring of th

useful output; and (3) the output system, which involves

JPEs(

State Key Laboratory of Bioelectronics (Chi

Science and Medical Engineering, Southe

China. E-mail: zyl22@cam.ac.uk; xuewang@

Cite this: Analyst, 2013, 138, 4427

Received 4th March 2013Accepted 8th May 2013

Analyst

MINIREVIEW

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article OnlineView Journal | View IssueNortheast University and thenjoined the State Key Laboratory

oUrctng

This journal is The Royal Society ofgnitive components, such asls, are an essential tool in thee parameters concerned with

amplication and display of the output in an appropriateformat.1 A transducer could convert the variation of the bio-logical sensing element into a measurable signal, such as anelectronic, photonic, thermal or mass signal.2,3 Based on thedierent ways of delivering signals, transducers can be catego-rized as electrochemical, eld-eect transistor (FET), optical,mechanical, piezoelectric, surface acoustic wave, and thermal

ian-Ling Wang is currently ah.D. candidate of Biomedicalngineering, Southeast Univer-ity. She obtained her M.S.Analytical Chemistry) from

Guihua Chen received a BS inScience and Technology of Elec-tronic Information at SichuanUniversity of Science & Engi-neering. She is currently an M.S.candidate in the School of Bio-logical Science and Medical

en-Shiung Wu Lab), School of Biological

ast University, Nanjing 210096, P. R.

seu.edu.cnscaled biosensors for disease bio-m

pathological cells and viruses, mo

prospects of biosensors in relevan

Introduction

Despite outstanding progress in the eld of biomedicine,signicant challenges remain in translating biomedicalknowledge of disease markers into clinically relevant devicesthat could be used as diagnostic or monitoring tools for diseasemanagement. Developing eective analytical techniques fordisease markers, pathogenic bacteria, and virus detection is

DOI: 10.1039/c3an00438d

www.rsc.org/analystf Bioelectronics, Southeastniversity in 2011. Her currentesearch focuses on theonstruction of nano-biosenors,he design of bio-functionalizedanostructures and tumor-tar-eting imaging.

Chemistry 2013no-biosensors oer a number of advantages and in many respects

applications, which could be made as extremely exible devices,

speediness, excellent selectivity and high sensitivity. This minireview

the latest years on the advances in biomedical applications of nano-

arking and detection, especially in bio-imaging and the diagnosis of

itoring pathogenic bacteria, thus providing insight into the future

clinical applications.

physiological or biochemical processes. Biosensors forbiomedical applications can be coupled to a physico-chemicaltransducer that converts this recognition into a detectableoutput signal,1 and provide a comprehensive review of estab-lished, cutting-edge and future trends in biomedical sensorsand their applications. Biosensors are typically comprised ofthree components: (1) the detector, which identies the stim-ulus; (2) the transducer, which converts this stimulus into aEngineering at SoutheastUniversity. Her interest is thefabrication of biosensors basedon nanomaterials for the detec-tion of GSH, NADH, glucose andpathogenic bacteria.

Analyst, 2013, 138, 44274435 | 4427

types,4,5 and others. According to recognitive components, thereare three classes of biosensors: molecular biosensors (i.e., basedon antibodies, nucleic acids, enzymes or ion channels),6,7

cellular biosensors, and tissue biosensors. As far as the sensitiveelements are concerned, the former are immobilized biologicalcomponents and the latter two are based on the organismsthemselves. Nowadays, with the rapid development of nano-technologies for biomedicine, biosensors based on novelnanomaterials and nanostructures are designed not only tosensitively respond to target molecules, but also to detectunexpected molecules. Nano-scaled biosensors have oered apowerful tool for analysis of the cellular microenvironment, aswell as the early diagnosis of pathological tissues.

As is well-known, nanomaterials have sizes ranging from a fewnanometers up to several hundred nanometers, comparable tomany biological macromolecules such as antibodies, enzymes,viruses and DNA plasmids. Materials in this size range exhibitinteresting physical properties, distinct from both the molecularand bulk scales, presenting new opportunities for biomedicalresearch and applications. The emerging eld of nano-

biotechnology bridges the physical sciences with biologicalsciences via chemical methods in developing novel tools andplatforms for understanding biological systems and diseasediagnosis and treatment.810 The above-mentioned bio-macromolecules are usually biomarkers as indicators of a bio-logical state or condition. In particular, the disease biomarker(i.e., proteins and protein fragments, DNA/RNA, or specic smallmolecules secreted by abnormal cells) is a molecular signatureof the physiological state of a disease at a specic time and istherefore a key for the early detection and accurate diagnosis ofdisease.11,12 Disease biomarkers also oer information on theunderlying mechanism of the initiation of a disease and ulti-mately provide powerful tools to precisely dene disease statesand treat the disease early enough.13,14 Thus, the fabrication ofbiosensors for disease biomarkers will be essential to detectaberrant genes or proteins at ultralow levels, as many biomarkersare present at minute concentrations during early disease pha-ses. In the meantime, nano-scaled biosensors could be moreportable and scalable for point-of-care sample analysis and real-time diagnosis.

Dr. Hui Jiang is an associateprofessor of Biomedical Engi-neering, Southeast University. Heobtained his Ph.D. (Chemistry)from the University of Scienceand Technology of China in 2005.Aer a postdoctoral fellowship inNanjing University, he joined theState Key Laboratory of Bio-electronics, Southeast Universityin 2007. His research focuses onthe nano-based electro-chemiluminescent biosensors andthe electron transfer behaviors of

Analyst Minireview

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article Onlinethe micro-nano interface.

Dr. Zhi-Yong Li is the Professorof Biomedical Engineering ofSoutheast University. He has aBSc degree in Structural Engi-neering and a Ph.D. in Biomed-ical Engineering. Between 2004and 2010, he worked as aResearch Fellow and then SeniorResearch Fellow at the Univer-sity of Cambridge, UK. He hasbeen a Fellow of Wolfson CollegeCambridge since 2005. Hisresearch interests include

cardiovascular biomechanics, image-based computationalmodeling, poroelastic theory and the mechanical behaviour ofdiseased arteries.4428 | Analyst, 2013, 138, 44274435We believe a review of the current advances in nano-scaledbiosensors for biomedical applications, discussing the latestdevelopments in the eld, is pertinent to the future directions ofrelevant biosensors. In recent years, similar articles have beenpublished. Wangs group has focused on constructing dierentkinds of nanostructure/nano-interfaces as biosensing platformsfor the selective determination and highly sensitive detection ofcancer cells, the rapid diagnosis of multidrug resistance incancer, and the evaluation of oxidative stress of tumor cells.Among the researches of biosensors based on the nanostructure/nano-interface, diverse nanomaterials or nanocomposites (i.e.,TiO2carbon nanotubes (CNT),15 gold nanoparticles,16 nano-TiO2ITO,17 gold nanoparticlespolylactide nanobers,18 andb-cyclodextrinmulti-walled carbon nanotubes19) were applied tofabricate new electrochemical cell sensors for determinatingcancer cells. Zhang et al.20 manufactured an electrochemicalsensor based on carbon nanotubesdrug supramolecular

Dr. Xuemei Wang is currently afull professor of BiomedicalEngineering, Southeast Univer-sity. She obtained her Ph.D. inChemistry from Nanjing Univer-sity, China in 1994 and becamea lecturer in Nanjing Universityin 1995. She was an Alexandervon Humboldt Fellow in theChemistry Department, Univer-sity of Saarland, Germany,before she joined the State KeyLaboratory of Bioelectronics,

Southeast University in 1998. Her research focuses on bio-electronics and biosensors, biomaterials, targeted drug deliverysystems and nanomedicine, bio-imaging and highly sensitivediagnosis strategies for cancers and related pathogenic bacteria.This journal is The Royal Society of Chemistry 2013

dierent types of spectroscopy, such as absorption, uores-

prostate, and that this disruption was detectable withMRI. Wanget al.32 recently impregnated gold nanoclusters (GNC) ontoreduced graphene oxide (RGO) nanosheets (GNCRGO) for switransport of anticancer drugs such as doxorubicin in hep-atocarcinoma (HepG2) cells (Fig. 1). GNCRGO nanocomposites,with excellent uorescence and surface enhanced Raman spec-troscopy characteristics, allow clear imaging of cellularmorphology and edges, indicating promising prospects forsimultaneous cellular imaging and acting as drug carriers. Thedesign of the GNCRGO novel nanostructure oers a multimodalplatform for targeting, detection, and oncotherapy. Furthermore,in vivo uorescence imaging of tumorsmay oer the possibility of

Minireview Analyst

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article Onlinecence, phosphorescence, Raman, Surface-enhanced Ramanscattering (SERS), refraction, and dispersion spectrometry.30 Inaddition, these spectroscopic methods can all measuredierent properties, such as energy, polarization, amplitude,decay time, and/or phase. Amplitude is the most commonlymeasured as it can easily realize the quanticational detectionof the specic analyte.30 This part focuses on optical nano-biosensors, and mainly about new strategies for the bio-imaging and diagnosis of pathological cells.

The early-stage detection of certain cancer cells in vivo isdicult since these cells generally do not metastasize.24 As such,imaging has become an indispensable tool in cancer clinicaltrials and medical practice, and there have been signicantadvances in the development of in vivo techniques of cancer cellimaging. For example, iron oxide particles have been used toidentify lymph node metastases in male prostate cancer.31 Thisresearch group found that the distribution of the iron oxideparticles was disrupted by malignant tumors present in thenanocomposites, providing a new strategy for the diagnosis ofmultidrug resistance in cancer. Chang et al.21 explored a facilestrategy to assess the oxidative stress elicited by H2O2 released bynormal or abnormal cells using a sensitive biosensor based onRGOAuPTBO (poly(toluidine blue O)) nanostructures, whichopened a novel avenue for the early diagnosis of cancer. Sub-ramanian et al. designed highly sensitive biosensors based oncarbon nanotube eld-eect transistor arrays to detect very smallamounts of genomic DNA from pathogenic bacteria.22 Tang'sgroup described the fabrication of carbon nanotube thin lmbiosensors for label-free and real-time electrical detection ofwhole viruses in a biocompatible buer solution.23 This minire-view discusses literature published in the latest years on theadvances in biosensors for biomedicine applications. Mean-while, we will divide this review into three main parts describingthe advances in: bio-imaging and the diagnosis of pathologicalcells, monitoring pathogenic bacteria, and the bio-analysis ofviruses based on nano-scaled biosensors.

New strategies for bio-imaging anddiagnosis of pathological cells

The identication and quantication of numerous biomarkersis required for the diagnosis, monitoring, and prognostic eval-uation of complex diseases such as cancer. Currently, 60% ofpatients diagnosed with breast, colon, lung, or ovarian canceralready have cell metastases forming in other locations of theirbody.2429 The development of eective diagnostic tools to detectthese cells has been dicult due to the low number of circu-lating cancer cells and the lack of suitable markers to identifythem. However, in vivo and in vitro applications of nano-scaledbiosensors based on an intelligent nano-interface or nano-structure may be used to increase the selectivity and resolutionand to make such diagnoses possible. The type of biosensorbased on the dierent kinds of functionalized materials hasbeen emerging in biomedicine elds. Especially, the biosensorbased on the optical signal, an optical biosensor, is the mostdiverse class of biosensors because they can be used for manyThis journal is The Royal Society of Chemistry 2013direct bio-imaging of tumors for the precise diagnosis of cancerand monitoring of the treatment process.33,34 For instance, theability to track the presence of uorescent nanoparticles in vivooers signicant improvements in the detection, diagnosis, andtreatment of diseases.

Surface-modied quantum dots (QDs) with bio-molecules (i.e.peptides, antibodies, nucleic acids, or small-molecule ligands34)can be used as in vivo imaging tools capable of binding to specictargets.35 Recently, this technology has been used to imagetumors in vivo.34 QDs have also been used for cell and tissuelabeling,36 long-term cell tracking, and multicolor cellimaging.37 Using nano-construction approaches, Nie and hiscolleagues realized the delivery of short-interfering RNA (siRNA)and intracellular imaging by designing multifunctional nano-particles based on the use of semiconductor QDs and proton-absorbing polymeric coatings (proton sponges) (Fig. 2).38 Thisspecial nano-design addressed long-standing barriers in siRNAdelivery such as cellular penetration, endosomal release, carrierunpacking, and intracellular transport. These results demon-strated the dramatic improvement in gene silencing eciency by1020-fold and the simultaneous reduction in cellular toxicity by56-fold, when compared directly with existing transfectionagents for MDA-MB-231 cells. The QD-siRNA nanoparticles, asdual-modality optical and electron-microscopy probes, shoul-dered real-time tracking and ultrastructural localization of QDsduring delivery and transfection. Despite their success, a numberof challenges remain for using QDs for in vivo applications. Oneof these challenges is that the core material for most QDs is aheavy metal that is toxic to cells, and accordingly, research iscurrently underway to produce alternatives with excellentbiocompatibility. Therefore, it may one day be possible to usenovel promising biocompatible nanoprobes to detect, diagnose,and treat diseases in a minimally invasive manner.

Fig. 1 Schematic illustration of gold nanoclusters and graphene nano-composites for drug delivery and imaging of cancer cells. (Image reproduced fromref. 32 with permission. Copyright 2011, John Wiley & Sons.)Analyst, 2013, 138, 44274435 | 4429

that the self-bio-imaging strategy has opened up promisingopportunities for biomedical applications requiring the specicand sensitive imaging of tumors without direct injection ofvectorized nanoparticles or molecular uorescent probes(Fig. 3). These bio-imaging techniques are examples of non-invasive cancer imaging tools that may be enhanced to includeadditional cancer cell types with future research.

Biosensors for monitoring pathogenicbacteria

Infectious disease, called communicable disease because of itsability to be transmitted from one person to another and alsosometimes from one species to another, e.g. u (inuenza), is aclinically evident disease resulting from the presence of a

Analyst Minireview

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article OnlineTo overcome the limited practical biomedical applications ofQDs due to their inherent cytotoxicity and self-aggregationinside living cells,39 uorescent nanoclusters (NCs) havereceived great attention and have been intensively studiedbecause of their superior properties such as their very strong,discrete, size-dependent emission,4042 low toxicity and highbiocompatibility. Thus, Au and Ag NCs are attractive candidatesfor making the smallest possible labels with strong oscillatorstrengths. Lately, much eort has been dedicated to the study ofuorescent Au NCs.4346 Au NCs carry quantum-mechanicalproperties when their sizes are comparable to or smaller thanthe Fermi wavelength (ca. 1 nm) of conductive electrons.47 Inaddition, in situ uorescent bio-imaging is also of great signif-icance for visualizing the expression and activity of particularmolecules, cells, and biological processes that inuence thebehavior of tumors and/or their responsiveness to therapeuticdrugs.48 In this case, Wang's group has realized the in vivo self-bio-imaging of tumors through in situ biosynthesized uores-cent gold nanoclusters,49 and has recently been developingnovel imaging techniques for early disease diagnoses based onother novel-function nanomaterials by the specicity of tumorsites or microenvironments. Experimental results indicated

Fig. 2 Rational design of proton-sponge coated quantum dots and their use as amultifunctional nanoscale carrier for siRNA delivery and intracellular imaging. (a)Chemical modication of polymer-encapsulated QDs to introduce tertiary aminegroups, and adsorption of siRNA on the particle surface by electrostatic interac-tions. (b) Schematic diagram showing the steps of siRNA-QD in membranebinding, cellular entry, endosomal escape, capturing by RNA binding proteins,loading to RNA-induced silencing complexes (RISC), and target degradation. (c)Schematic illustration of the proton-sponge eect showing the involvement ofthe membrane protein ATPase (proton pump), osmotic pressure build-up, andorganelle swelling and rupture. (Image reproduced from ref. 38 with permission.Copyright 2008, American Chemical Society.)

4430 | Analyst, 2013, 138, 44274435pathogenic agent which can either be a pathogenic virus orbacteria or fungi or a parasite.50 While infectious diseases caninitiate in a localized region, they can spread rapidly at anymoment due to the ease of traveling from one part of the worldto the next. Therefore, the rapid and accurate detection of traceamounts of organisms, such as pathogenic bacteria, is impor-tant for food and water safety, clinical diagnosis, and theprevention of accidental outbreaks. In the case of intentionalterrorist acts, early detection of trace amounts of pathogenicmicro-organisms is critical. Even more, pathogenic bacteria canlead to an infectious disease.51,52 Traditional methods not onlyrequire well-trained experts and involve long assay times butalso need cumbersome steps and expensive spent.5355 There-fore, new strategies for detecting potential pathogenic bacteriabecome more and more signicant and urgent.

Biosensors, which combine bio-recognition elements andsignal transducers to detect target compounds, have beenproved to be promising analytical devices for the detection ofpathogenic bacteria.56,57 In the recent years, a series of biosen-sors have been produced to detect pathogenic bacteria based onelectronic, colorimetric, uorescent, and electrochemical tech-niques. For example, McAlpine et al. designed antimicrobialpeptide-functionalized micro-capacitive electrode arrays todetect bacterial infectious agents by an electronic strategy.58

Rotello and his colleagues fabricated a colorimetric biosensorby conjugating an enzyme to Au nanoparticles for bacterialsensing.59 Cheng et al. developed an electrochemical biosensor

Fig. 3 Schematic illustration of in situ biosynthesis of gold nanoclusters in cancercells and tumor imaging. (Image reproduced from ref. 49 with permission.Copyright 2013, Nature Publishing Group.)This journal is The Royal Society of Chemistry 2013

chemical techniques.79,85,86

Minireview Analyst

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article Onlinewith a cell membrane-like nanostructure to detect Escherichiacoli (E. coli) bacteria.60 Hewlett's group developed an immuno-assay based on polystyrene NPs loaded with Eu(III) b-diketonechelates for the detection of anthrax protective agent (PA).61

Fang et al. demonstrated another Au NP immunoassay readouttechnique for the easy screening of Salmonella.62 All eorts havedemonstrated that biosensors are simper, faster and moresensitive for bacterial sensing and do not need professionaloperational skills.

Thereinto, aptamer-based biosensors have been playing animportant role in the detection of pathogenic bacteria inbiomedical elds. Aptamers, which are a kind of syntheticoligonucleotide, can bind a certain target with extremely highsensitivity and specicity.63,64 Ye and his co-workers65 fabricateda surface plasmon resonance (SPR) immunosensor based onaptamers for the detection of E. coli O157:H7. This method canavoid the SPR defects of bacteria size, which in turn increasedthe sensitivity of the biosensor. Their results showed that theSPR immunosensor can detect E. coli O157:H7 cells in a rangefrom 3.0 104 to 3.0 108 CFU mL1 with a detection limitof 3.0 104 CFU mL1. Ju's group66 developed a rapid andsensitive electrochemical biosensor based on aptamers for thedirect detection of E. coli O111. The biosensor they proposedcould directly and sensitively detect E. coli O111 in phosphatebuer with the detection limit of 112 CFU mL1, and could beapplied for detection in milk. Zhang et al. developed a uores-cent bio-barcoded DNA assay for the rapid detection of theSalmonella enteritidis gene, based on two NPs: magneticnanoparticles (MNP) and gold nanoparticles (Au NP).67 The AuNPs are conjugated with the 1st target-specic DNA probe whichcan recognize the target gene, and a uorescein-labeled barcodeDNA. The MNPs were coated with the 2nd target-specic DNAprobe. Upon mixing with the target gene, a sandwich structureis formed followed by magnetic separation of the sandwichstructure (Fig. 4). The barcode DNA from the Au NPs is releasedby heating the mixture, and the released barcode DNA ismeasured by uorescence with a detection limit of 1 ng mL1.The Au NPs here contribute to signal amplication by carrying alarge amount of barcode DNA per DNA probe binding event andthe MNPs act as a separator and pre-concentrator. Thesebiosensors have high sensitivity and stability, but eachbiosensor can only detect one kind of bacteria.

Though some strategies have been improved in the situationof pathogenic bacteria detection, the simple, easy-to-use, cheapand easy-to-manufacture strategy is still the hot topic. Kim'sgroup developed a simple strip-type chemiluminescent immu-nosensor to detect E. coli O157:H7 (Fig. 5A).68 The biosensor ishighly sensitive with a low detection limit of 1.0 103CFU mL1 and can be successfully used to detect E. coliO157:H7 in a range of 1.1 103 to 1.1 107 CFU mL1 within16 min. Zakir Hossain et al.69 reported a paper-based stripbiosensor for the detection E. coli O157:H7. First, either5-bromo-4-chloro-3-indolyl-b-D-glucuronide sodium salt orchlorophenol red b-galactopyranoside (CPRG) (color reaction orCR zone) and FeCl3 are entrapped within solgel-derived silicamaterials in the two dashed regions on a paper strip. Then thehydrophobic barrier composed of MSQ is layered at the top ofThis journal is The Royal Society of Chemistry 2013To date, nanoparticles (NPs) of various types have beenprimarily studied and have shown great promise for the rapidand accurate identication of viruses. A few platforms based onmicro/nanomaterials, including whispering-gallery micro-lasers,87 opto-uidic micro-ring resonators,88 organic FETs,89

and SPR,90 have been reported with the ability to detect a singleinuenza A virus in a buered or serum solution, whichoutlines the immense potential of whole-virus detection tech-niques. Ray and co-workers screened and quantied thesequence-specic hepatitis C virus (HCV) RNA using the non-linear optical properties of gold nanoparticles (Au NP) by Hyperthe sensing zone. When the sensor was dipped into a cell lysate,the color appearance in the CR zone could be indicative of thepresence of bacteria. Using this paper biosensor could detectE. coli BL21 (LOD 20 CFU mL1) and E. coli O157:H7 (LOD5 CFU mL1) within 30 min without culturing, the resultshowed that the biosensor is rapid, selective, and ultra-sensi-tive. All of these proposals were described in Fig. 5B. The paperbased biosensors are rapid and intuitive for users, however, thesensitivity may be unsatisfactory, and some other strategiesshould be induced to make the paper based biosensors moreuseful.

Bioanalysis of virus based on nano-biosensors

Many transitions in healthcare systems have been witnessedaround the globe. The phenomenon of infectious diseases, inpart attributed to the emergence of the human immunode-ciency virus (HIV)-AIDS epidemics, has been illustrating thedynamic nature of infectious diseases and exemplies thevulnerability of populations to emerging diseases, as seenduring the Severe Acute Respiratory Syndrome (SARS)epidemics of 2003 and the H1N1 inuenza pandemic of 2009.Rapid and accurate identication of viruses is critical to bothmedical diagnosis and bio-defense,23 and has been a seriouspublic health, homeland security, and armed forces issue.70 Acritical aspect of recognizing and controlling future epidemicswill be the development of rapid and sensitive diagnostictechniques that can be quickly deployed at multiple sites.71

Traditional detection methods such as cell culturing, enzyme-linked immunosorbent assays (ELISA), and polymerase chainreaction (PCR) are not readily compatible with point-of-care usewithout the existence of extensive infrastructure.72,73 Therefore,highly sensitive/specic, compact, fast, and easy-to-use virusdetections and diagnostics are needed to prevent further spreadat the onset of a viral epidemic. A variety of novel sensingtechniques has been developed for this purpose, the majority ofwhich identify and characterize viruses through the detection oftheir signature proteins7479 or DNA.7984 Thereinto, functionalbiosensors are formed by immobilized bio-receptors onto atransducer, which have high specicity for the target analyte.The biosensor can realize the ecient, specic and rapid bio-analysis of viruses by multimodes based on nanostructures, i.e.,surface plasmon resonance (SPR), ber optics, acoustic wavetechnologies, quartz crystal microbalance (QCM) and electro-Analyst, 2013, 138, 44274435 | 4431

Fig. 4 Schematic of the bio-barcode assay: (A) formation of MNP-2nd DNA probe/trelease. (Image reproduced from ref. 67 with permission. Copyright 2012, John wil

Fig. 5 (A) Schematic illustration of the working principle of chemiluminescentimmunosensors. (Image reproduced from ref. 68 with permission. Copyright2012, Taylor & Francis Group.) (B) The paper based strip biosensor nano-biosensor. (Image reproduced from ref. 69 with permission. Copyright 2012,Springer.)

4432 | Analyst, 2013, 138, 44274435

Analyst Minireview

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article OnlineRayleigh scattering (HRS) technique, and accomplished thedirect HCV RNA-based test to detect its presence at an earlystage of infection.91 The assay is based on the fact that double-and single-stranded oligonucleotides have dierent electro-static properties. A uorescent Raman dye rhodamine 6G tag-ged ssRNA was adsorbed onto the Au NPs. Fluorescencequenching of the dye and the enhancement of resonant Ramanscattering from the dye were observed. Upon binding to thetarget RNA, the duplex structure of the double stranded (ds)

arget DNA/1st DNA probe-Au-NPs-barcode DNA; (B) barcode DNA separation andey & Sons.)RNA is formed. Electrostatic repulsion between the ds RNA andthe NP causes the ds RNA not to adsorb onto the Au NPs and theuorescence of the dye persists. As soon as the ds RNA isseparated from the Au NPs, a second eect is observed which isthe aggregation of the Au NPs. This aggregation is evidenced bytransmission electron microscopy (TEM) and further conrmedby colorimetric studies. The aggregation causes an increase insize resulting in an increase in the HRS intensity, thereby con-rming the detection of HCV virus RNA. They used a similarHRS assay with Au nanorods for sensing sequence-specic HIV-1 virus DNA.92 Yanik et al.93 developed a label-free optouidicnanoplasmonic sensor for the sensitive and rapid detection ofintact viruses from biological media at clinically relevantconcentrations with little to no sample preparation (Fig. 6). Thesensing platform consisted of a suspended nanohole arraygrating that couples the normally incident light to surfaceplasmons electromagnetic waves trapped at the metaldielectric interface in coherence with collective electron oscil-lations and used antiviral immunoglobulins immobilized atthe sensor surface for specic capture of the virions to quantifyand identify virus concentrations. This could identify a broadrange of known and even previously unknown pathogens (i.e.,novel mutant strains). Such quantitative detection makes itpossible to detect not only the presence of the intact viruses in

This journal is The Royal Society of Chemistry 2013

Minireview Analyst

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article Onlinethe analyzed samples but also the intensity of the infectionprocess. Their results proved that this sensing platform enableslabel-free virus detection within a concentration window

Fig. 6 Three-dimensional renderings (not drawn to scale) and the experimentalmeasurements illustrate the detection scheme using optouidic nanoplasmonicbiosensors based on resonance transmissions due to the extraordinary lighttransmission eect. (a) Detection (immobilized with capturing antibody targetingthe vesicular stomatitis virus (VSV)) and control sensors (unfunctionalized) areshown. (b) VSV attaches only to the antibody immobilized sensor. (c) Noobservable shift is detected for the control sensor after the VSV incubation andwashing. (d) Accumulation of the VSV due to the capturing by the antibodies isexperimentally observed. (Image reproduced from ref. 93 with permission.Copyright 2010, American Chemical Society.)ranging from that needed for clinical testing to drug screening.The above-mentioned biosensors based on nanomaterials openup opportunities for the detection of a broad range of patho-gens in typical biomedical elds.

Conclusions and future prospects

The recent progress of biosensors based on micro- and nano-scaled technologies has been emerging in biomedical elds,and shows signicant promise in the detection, analysis andmonitoring of the parameters concerned with physiological orbiochemical processes. Highly sensitive biosensors could beused for the early detection of prognostic biomarker levels,which can distinguish between the favorable and unfavorableoutcomes of diseases (especially tumors) and guide furtherdisease treatment. Based on various kinds of functionalizednanostructures or nano-interfaces, the nanoscaled biosensorscould be readily utilized for point-of-care sample analysis andreal-time diagnosis in a clinical area. A growing number ofrecent researches have explored the utility of nano-scaledbiosensors for biomedical applications, particularly in light offabricating biosensors that are portable, cheap, and highlysensitive that can be used for diagnosing diseases or moni-toring their progression in medicine. However, the currentsensitivity of nano-biosensors can be only obtained underhighly optimized conditions in a laboratory. Mostly, nano-biosensors determine analytes in clinical samples, seldom with

11 J. A. Ludwig and J. N. Weinstein, Nat. Rev. Cancer, 2005, 5,

B. R. Ilic, D. J. Mooney, M. A. Reed and T. M. Fahmy, Nat.

This journal is The Royal Society of Chemistry 2013Nanotechnol., 2010, 5, 138142.15 Q. Shen, S. You, S. Park, H. Jiang, D. Guo, B. Chen and

X. Wang, Electroanalysis, 2008, 20, 25262530.16 F. He, Q. Shen, H. Jiang, J. Zhou, J. Cheng, D. Guo, Q. Li,

X. Wang, D. Fu and B. Chen, Talanta, 2009, 77, 10091014.17 X. Wu, H. Jiang, Y. Zhou, J. Li, C. Wu, C. Wu, B. Chen and

X. Wang, Electrochem. Commun., 2010, 12, 962965.18 X. Wu, H. Jiang, J. Zheng, X. Wang, Z. Gu and C. Chen,

J. Electroanal. Chem., 2011, 656, 174178.19 J. Zhao, J. S. Jin, C. H. Wu, H. Jiang, Y. Y. Zhou, J. L. Zuo and

X. M. Wang, Analyst, 2010, 135, 29652969.845856.12 J. M. Nam, C. S. Thaxton and C. A. Mirkin, Science, 2003, 301,

18841886.13 M. Swierczewska, G. Liu, S. Lee and X. Chen, Chem. Soc. Rev.,

2012, 41, 26412655.14 E. Stern, A. Vacic, N. K. Rajan, J. M. Criscione, J. Park,in vivomonitoring. In addition, it is still essential to address thenanomaterialsbiomarkers interaction processes in dierentbiological microenvironments. When designing nano-scaledbiosensors towards robust signal amplication for biomedicalapplications, much attention must focus on integration ofmolecular recognition elements and the nanostructure/nano-interface for biomedical analysis. Much emphasis should be puton the strategies currently used to improve the performance ofbiosensors, the ecient status of nano-scaled biosensors forbiomedicine, and the trends and challenges envisaged for thenear future.

Acknowledgements

This work is supported by the National Basic Research Programof China (no. 2010CB732404), National Natural-Science Foun-dation of China (21175020, 90713023), and Suzhou Science &Technology Major Project (ZXY2012028).

References

1 A. F. Collings and F. Caruso, Rep. Prog. Phys., 1997, 60, 13971445.

2 X. Y. Wu, Y. Q. Chai, R. Yuan, H. L. Su and J. Han, Analyst,2013, 138, 10601066.

3 A. Gao, C. X. Tang, X. W. He and X. B. Yin, Analyst, 2013, 138,263268.

4 B. R. Higgins and H. H. Weetall, Appl. Biochem. Biotechnol.,1998, 73, 7980.

5 K. K. Jain, Nanobiotechnology in Molecular Diagnostics:Current Techniques and Application, Horizon Bioscience,UK, 2006.

6 H. Jiang and X. M. Wang, Analyst, 2012, 137, 25822587.7 H. C. Chang, X. J. Wu, C. Y. Wu, Y. Chen, H. Jiang andX. M. Wang, Analyst, 2011, 136, 27352740.

8 G. M. Whitesides, Nat. Biotechnol., 2003, 21, 11611165.9 C. R. Lowe, Curr. Opin. Chem. Biol., 2000, 10, 428434.10 L. Wang, W. Zhao and W. Tan, Nano Res., 2008, 1, 99115.Analyst, 2013, 138, 44274435 | 4433

Analyst Minireview

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article Online20 H. Zhang, H. Jiang, F. Sun, H. Wang, J. Zhao, B. Chen andX. Wang, Biosens. Bioelectron., 2011, 26, 33613366.

21 H. Chang, X. Wang, K. Shiu, Y. Zhu, J. Wang, Q. Li, B. Chenand H. Jiang, Biosens. Bioelectron., 2013, 41, 789794.

22 S. Subramanian, K. H. Aschenbach, J. P. Evangelista,M. B. Najjar, W. X. Song and R. D. Gomez, Biosens.Bioelectron., 2012, 32, 6975.

23 H. S. Mandal, Z. Su, A. Ward and X. Tang, Theranostics, 2012,2, 251257.

24 P. Grodzinski, M. Silver and L. K. Molnar, Expert Rev. Mol.Diagn., 2006, 6, 307318.

25 A. Han, L. Yang and A. B. Frazier, Clin. Cancer Res., 2007, 13,139143.

26 I. Desitter, B. S. Guerrouahen, N. Benali-Furet, J. Wechsler,P. A. Janne, Y. Kuang, M. Yanagita, L. Wang, J. A. Berkowitz,R. J. Distel and Y. E. Cayre, Anticancer Res., 2011, 31, 427442.

27 D. A. Smirnov, D. R. Zweitzig, B. W. Foulk, M. C. Miller,G. V. Doyle, K. J. Pienta, N. J. Meropol, L. M. Weiner,S. J. Cohen, J. G. Moreno, M. C. Connelly,L. W. M. M. Terstappen and S. M. O'Hara, Cancer Res., 2005,65, 49934997.

28 L. Bonmassar, E. Fossile, A. Scoppola, G. Grazizanik,S. P. Prete, V. Formica, D. Cappelletti, L. D. Vecchis,A. Cardillo, F. Concolino, S. D'Atri, A. Bakdyzzum,F. Torino, P. Caporaso, J. W. Greiner, E. Bonmassar,M. Roselli and A. Aquino, Anticancer Res., 2010, 30, 47214730.

29 G. Colloca, Cancer Treat. Rev., 2012, 38, 10201026.30 T. Kubik, K. Bogunia-Kubik and M. Sugisaka, Curr. Pharm.

Biotechnol., 2005, 6, 1733.31 L. Tang, X. Yang, L. W. Dobrucki, I. Chaudhury, Q. Yin,

C. Yao, S. Lezmi, W. G. Helferich, T. M. Fan and J. Cheng,Angew. Chem., 2012, 124, 1289312898.

32 C. Wang, J. Li, C. Amatore, Y. Chen, H. Jiang and X. Wang,Angew. Chem., Int. Ed., 2011, 50, 1164411648.

33 S. Jiang, M. K. Gnanasammandhan and Y. Zhang, J. R. Soc.Interface, 2010, 7, 318.

34 X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung andS. M. Nie, Nat. Biotechnol., 2004, 22, 969976.

35 W. C. Chan and S. Nie, Science, 1998, 281, 20162018.36 A. M. Smith, H. W. Duan, A. M. Mohs and S. M. Nie, Adv.

Drug Delivery Rev., 2008, 60, 12261240.37 J. H. Gao, X. Y. Chen and Z. Cheng, Curr. Top. Med. Chem.,

2010, 10, 11471157.38 M. V. Yezhelyev, L. Qi, R. M. O'Regan, S. Nie and X. Gao, J.

Am. Chem. Soc., 2008, 130, 90069012.39 A. A. Bhirde, V. Patel, J. Gavard, G. Zhang, A. A. Sousa,

A. Masedunskas, R. D. Leapman, R. Weigert, J. S. Gutkindand J. F. Rusling, ACS Nano, 2009, 3, 307316.

40 M. Zhang, Y.-Q. Dang, T.-Y. Liu, H.-W. Li, Y. Wu, Q. Li,K. Wang and B. Zou, J. Phys. Chem. C, 2013, 117, 639647.

41 M. Harb, F. Rabilloud and D. Simon, Chem. Phys. Lett., 2009,476, 186190.

42 F. Qu, N. B. Li and H. Q. Luo, Anal. Chem., 2012, 84, 1037310379.

43 J. M. Abad, L. E. Sendroiu, M. Gass, A. Bleloch, A. J. Mills andD. J. Schirin, J. Am. Chem. Soc., 2007, 129, 1293212933.4434 | Analyst, 2013, 138, 4427443544 A. Dass, A. Stevenson, G. R. Dubay, J. B. Tracy andR. W. Murray, J. Am. Chem. Soc., 2008, 130, 59405946.

45 M. Eichelbaum, B. E. Schmidt, H. Ibrahim andK. Rademann, Nanotechnology, 2007, 18, 355702.

46 P. D. Jadzinsky, G. Calero, C. J. Ackerson, D. A. Bushnell andR. D. Kornberg, Science, 2007, 318, 430433.

47 C. C. Huang, Z. Yang, K. H. Lee and H. T. Chang, Angew.Chem., Int. Ed., 2007, 46, 68246828.

48 R. Weissleder and M. J. Pittet, Nature, 2008, 452, 580589.49 J. Wang, G. Zhang, Q. Li, H. Jiang, C. Liu, C. Amatore and

X. Wang, Sci. Rep., 2013, 3, 1157.50 P. Tallury, A. Malhotra, L. M. Byrne and S. Santra, Adv. Drug

Delivery Rev., 2010, 62, 424437.51 Y. X. Wang, Z. Z. Ye and Y. B. Ying, Sensors, 2012, 12, 3449

3471.52 D. Jacob, U. Sauer, R. Housley, C. Washington, K. Sannes-

Lowery, D. J. Ecker, R. Sampath and R. Grunow, PLoS One,2012, 7, 3992839934.

53 X. Y. Z. Karsunke, N. Reinhard and S. Michael, Anal. Bioanal.Chem., 2009, 395, 16231630.

54 X. F. Guo, A. Kulkarni, A. Doepke, H. B. Halsall, S. Iyer andW. R. Heineman, Anal. Chem., 2012, 84, 241246.

55 A. Ruchi, A. Satlewal, M. Chaudhary, A. Verma, R. Singh,A. K. Verma, R. Kumar and K. P. Singh, J. Microbiol.Biotechnol., 2012, 22, 849855.

56 Y. X. Wang, Z. Z. Ye, C. Y Si and Y. B. Ying, Chin. J. Anal.Chem., 2012, 40, 634642.

57 C. Y. Wen, J. Hu, Z. L. Zhang, Z. Q. Tian, G. P. Ou, Y. L. Liao,Y. Li, M. Xie, Z. Y. Sun and D. W. Pang, Anal. Chem., 2013, 85,12231230.

58 M. S. Mannoor, S. Zhang, A. J. Link andM. C. McAlpine, Proc.Natl. Acad. Sci. U. S. A., 2010, 107, 1920719212.

59 O. R. Miranda, X. N. Li, L. Garcia-Gonzalez, Z. J. Zhu, B. Yan,U. H. F. Bunz and V. M. Rotello, J. Am. Chem. Soc., 2011, 133,96509653.

60 M. S. Cheng, S. H. Lau, V. T. Chow and C. S. Toh, Environ. Sci.Technol., 2011, 45, 64536459.

61 S. Tang, M. Moayeri, Z. Chen, H. Harma, J. Zhao, H. Hu,R. H. Purcell, S. H. Leppla and I. K. Hewlett, Clin. VaccineImmunol., 2009, 16, 408413.

62 S. B. Fang, W. Y. Tseng, H. C. Lee, C. K. Tsai, J. T. Huang andS. Y. Hou, J. Microbiol. Methods, 2009, 77, 225228.

63 C. Tuerk and L. Gold, Science, 1990, 249, 505510.64 A. D. Ellington and J. W. Szostak, Nature, 1990, 346, 818822.65 Y. X. Wang, Z. Z. Ye, C. Y. Si and Y. B. Ying, Sensors, 2011, 11,

27282739.66 C. H. Luo, Y. N. Lei, L. Yan, T. X. Yu, Q. Li, D. C. Zhang,

S. J. Ding and H. X. Ju, Electroanalysis, 2012, 24, 11861191.67 D. Zhang, D. J. Carr and E. C. Alocilja, Biosens. Bioelectron.,

2009, 24, 13771381.68 S. J. Park, J. H. Min and Y. K. Kim, Int. J. Environ. Anal. Chem.,

2012, 92, 655664.69 S. M. Zakir Hossain, C. Ozimok, C. Sicard, S. D. Aguirre,

M. M. Ali, Y. F. Li and J. D. Brennan, Anal. Bioanal. Chem.,2012, 403, 15671576.

70 A. A. Yanik, M. Huang, O. Kamohara, A. Artar, T. W. Geisbert,J. H. Connor and H. Altug, Nano Lett., 2010, 10, 49624969.This journal is The Royal Society of Chemistry 2013

71 D. Tseng, O. Mudanyali, C. Oztoprak, S. O. Isikman,I. Sencan, O. Yaglidere and A. Ozcan, Lab Chip, 2010, 10,17871792.

72 J. H. Henkel, S. W. Aberle, M. Kundi and T. Popow-Kraupp, J.Med. Virol., 1997, 53, 366371.

73 P. D. Bao, T. Q. Huang, X. M. Liu and T. Q. Wu, J. RamanSpectrosc., 2001, 32, 227230.

74 M. Abe, K. Murata, A. Kojima, Y. Ifuku, M. Shimizu, T. Atakaand K. Matsumoto, J. Phys. Chem., 2007, 111, 86678670.

75 Y. Long, Z. Zhang, X. Yan, J. Xing, K. Zhang, J.-X. Huang andW. Li, Anal. Chim. Acta, 2010, 665, 6368.

76 L. Su, R. Chen, Y. Li, Y. Chang, Y. Lee, C. Lee and C. Chou,Anal. Chem., 2010, 82, 37143718.

77 B. Zhang, X. Zhang, H. Yan, S. Xu, D. Tang and W. Fu,Biosens. Bioelectron., 2007, 23, 1925.

78 A. Palaniappan, W. H. Goh, J. N. Tey, I. P. M. Wijaya,S. M. Moochhala, B. Liedberg and S. G. Mhaisalkar,Biosens. Bioelectron., 2010, 25, 19891993.

79 L. M. Lechuga, J. Tamayo, M. Alvarez, L. G. Carrascosa,A. Yufera, R. Doldan, E. Peralas, A. Rueda, J. A. Plaza,K. Zinoviev, C. Domnguez, A. Zaballos, M. Moreno,C. Martnez-A, D. Wenn, N. Harris, C. Bringer, V. Bardinal,T. Camps, C. Vergnene`gre, C. Fontaine, V. Daz andA. Bernad, Sens. Actuators, B, 2006, 118, 210.

80 A. Star, E. Tu, J. Niemann, J. C. P. Gabriel, C. S. Joiner andC. Valcke, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 921926.

81 G. Giraud, H. Schulze, T. T. Bachmann, C. J. Campbell,A. R. Mount, P. Ghazal, M. R. Khondoker, S. W. J. Ember,

I. Ciani, C. Tlili, A. J. Walton, J. G. Terry and J. Crain,Chem. Phys. Lett., 2010, 484, 309314.

82 C. Chiu, H. Lee, C. Kuo and S. Gwo, Appl. Phys. Lett., 2008, 93,13.

83 J. Baur, C. Gondran, M. Holzinger, E. Defrancq, H. Perrotand S. Cosnier, Anal. Chem., 2010, 82, 10661072.

84 J. Zhang, H. P. Lang, F. Huber, A. Bietsch, W. Grange,U. Certa, R. Mckendry, H.-J. Guntherodt, M. Hegner andCh. Gerber, Nat. Nanotechnol., 2006, 1, 214220.

85 A. W. Wark, J. Lee, S. Kim, S. N. Faisal and H. J. Lee, J. Ind.Eng. Chem., 2010, 16, 169174.

86 J. Heo and S. Z. Hua, Sensors, 2009, 9, 44834502.87 F. Vollmer, S. Arnold and D. Keng, Proc. Natl. Acad. Sci. U. S.

A., 2008, 105, 2070120704.88 H. Zhu, I. M. White, J. D. Suter, M. Zourob and X. Fan,

Analyst, 2008, 133, 356360.89 M. D. Angione, S. Cotrone, M. Magliulo, A. Mallardi,

D. Altamura, C. Giannini, N. Cio, L. Sabbatini, E. Fratini,P. Baglioni, G. Scamarcio, G. Palazzo and L. Torsi, Proc.Natl. Acad. Sci. U. S. A., 2012, 109, 64296434.

90 S. Wang, X. Shana, U. Patela, X. Huang, J. Lu, J. Li andN. Tao, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 1602816032.

91 J. Grin, A. K. Singh, D. Senapati, E. Lee, K. Gaylor, J. Jones-Boone and P. C. Ray, Small, 2009, 5, 839845.

92 G. K. Darbha, U. S. Rai, A. K. Singh and P. C. Ray, Chem.Eur.J., 2008, 14, 38963903.

93 A. A. Yanik, M. Huang, O. Kamohara, A. Artar, T. W. Geisbert,J. H. Connor and H. Altug, Nano Lett., 2010, 10, 49624969.

Minireview Analyst

Publ

ished

on

09 M

ay 2

013.

Dow

nloa

ded

by U

nive

rsid

ade T

ecno

logi

ca F

eder

al d

o Pa

rana

on

25/0

3/20

15 1

2:48

:42.

View Article OnlineThis journal is The Royal Society of Chemistry 2013 Analyst, 2013, 138, 44274435 | 4435

Advances in nano-scaled biosensors for biomedical applicationsAdvances in nano-scaled biosensors for biomedical applicationsAdvances in nano-scaled biosensors for biomedical applicationsAdvances in nano-scaled biosensors for biomedical applicationsAdvances in nano-scaled biosensors for biomedical applicationsAdvances in nano-scaled biosensors for biomedical applicationsAdvances in nano-scaled biosensors for biomedical applications