DOI: 10.1002/chem.201300031

Selective, Sensitive, and Rapid Analysis with Lateral-Flow Assays Based onAntibody-Gated Dye-Delivery Systems: The Example of

Triacetone Triperoxide

Estela Climent,[a] Delia Grçninger,[b] Mandy Hecht,[b] M. Astrid Walter,[b]

Ram�n Mart�nez-M�Çez,*[a] Michael G. Weller,[b] F�lix Sancen�n,[a] Pedro Amor�s,[c] andKnut Rurack*[b]

Nanoscopic delivery systems have received tremendousattention in recent years because they can be designed froma large number of materials in a great variety of architec-tures, thus enabling precise tailoring and control of theirfunction in a desired location.[1] However, although theirpopularity in drug delivery or related biomedical and phar-maceutical applications is well documented,[2] their break-through in the area of analytical sciences is yet to come.[3]

The potential of such systems for analytical purposes isnonetheless immediately clear. Provided that the carriersystem is loaded with an indicator and the appropriate de-tection chemistry is implemented in a way that only theadvent of an analyte molecule can trigger delivery, such sys-tems can release a much larger number of indicators than

the number of analytes necessary to induce release, whichresults in amplified signaling. The key is thus to equip suchsystems with a selective gating mechanism that controls therelease of the measureable cargo. Few examples have beenreported in recent years that operate with specific gatingchemistries and target selected analytes in conventional sol-ution-based experiments.[4] A more general approach and itsimplementation within an actually applicable sensing formathas not been realized so far. Herein, we report the develop-ment of such a general approach by designing an antibody-gated dye-delivery system and incorporating it with a con-ventional test-strip-based lateral-flow assay, finally allowingfor small-molecule sensing at the ppb level in an easy-to-op-erate manner and an overall assay time of 2–10 min, whichis fast for a biochemical test.

The model analyte chosen was triacetone triperoxide(TATP). TATP is the prototype of a small-molecule analytethat is difficult to target with supramolecular recognitionchemistry because it lacks functional groups for classicalnoncovalent binding and is neither particularly polar norapolar. TATP is also a high-priority analyte because of itspopular use in improvised explosive devices (IEDs) forcriminal and terrorist activities.[5] Recent prevention strat-egies increasingly focus on the tracing of explosives, for in-stance, in sewage water to locate IED “bomb factories”,[6]

so potent on-site sensing systems for the condensed phaseare required. However, although various methods for lab-ACHTUNGTRENNUNGoratory-based TATP detection in the liquid phase are availa-ble, selective and sensitive portable sensing systems are veryscarce.[7] In addition, because most chemosensing systemsrely on the decomposition of TATP and the subsequent de-tection of one of its precursors, hydrogen peroxide,[8] thedirect detection of TATP without interference by other per-oxide-based explosives and H2O2 would be desirable fortracing TATP in environments in which cleaning, bleaching,and other domestic agents are used and could lead to un-wanted false-positive alarms.

Our approach for the specific detection of TATP in liquidsamples at trace levels with a portable, handheld, and rapid-ly responding device with straightforward operation is atest-strip-based assay with simple fluorescence readout.Test-strip assays for such purposes—especially if neutral

[a] Dr. E. Climent, Prof. R. Mart�nez-M�Çez, Dr. F. Sancen�nCentro de Reconocimienro Molecular yDesarrollo Tecnol�gico (IDM)Unidad mixta Universitat Polit�cnica de Val�nciaUniversitat de Val�ncia, Departamento de Qu�micaUniversidad Polit�cnica de ValenciaCamino de Vera s/n, 46022 Valencia (Spain)Fax: (+34) 96-387-93-49andCIBER de Bioingenier�aBiomateriales y Nanomedicina (CIBER-BBN)E-mail : rmaez@qim.upv.es

[b] D. Grçninger, M. Hecht, Dr. M. A. Walter, Dr. M. G. Weller,Dr. K. RurackAbteilung 1 Analytische Chemie, ReferenzmaterialienBAM Bundesanstalt f�r Materialforschung und -pr�fungRichard-Willst�tter-Strasse 11, 12489 Berlin (Germany)Fax: (+49)30-8104-1157E-mail : knut.rurack@bam.de

[c] Prof. P. Amor�sInstitut de Ci�ncia dels Materials (ICMUV)Universitat de Val�nciaP.O. Box 2085, 46071 Valencia (Spain)

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201300031. It contains details ofgeneral techniques, reagents employed, synthesis of precursors andsolids, and full characterization of compounds and solids, includingfigures of XRD and TEM analysis, porosimetry isotherms, antibody–antigen binding, lateral-flow assay protocols, detailed performancetesting, measurement uncertainties, and LODs.

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COMMUNICATION

small organic molecules unable to influence the optical orredox properties of indicator molecules to a significantextent are concerned—require the integration of signal amp-ACHTUNGTRENNUNGlification features. As mentioned above, chemically gateddelivery systems are particularly attractive in this regard.Based on our experience in the fields of bio-gated releasesystems[9] and gated hybrid materials for sensing applica-tions,[4] we designed the ensemble sketched in Figure 1. Or-dered mesoporous silica nanoparticles (MSNs)[10] are usedas the nanoscopic support with a defined void structure,high inner surface area, and flexible functionalization chem-istry. These MSNs are then loaded with the brightly fluores-cent indicator dye sulforhodamine B (to yield solid S0) andthe external surface is subsequently functionalized with asuitable hapten (S1). Finally, a TATP-selective polyclonalantibody (S1-AB) recently developed by us[11,12] serves as abulky stopper moiety, which is attached at the void openingsand efficiently closes them. The tailored relay chemistry thatconnects stopper and support is thus based on noncovalentantibody–antigen interactions and (ideally) only TATP asstimulus should be able to successfully compete with thehapten for the antibodys binding sites, to dislocate the stop-pers, to open the voids, and to release the entrapped indica-

tor. The last can be monitoredconveniently at excitationwavelengths between 500 and570 nm and emission wave-lengths between 550 and650 nm, which cover prominentexcitation sources and emissionfilter windows. The completepreparation and characteriza-tion procedures for the materi-als are given in the SupportingInformation.

An important feature whenaiming to use the cappedhybrid delivery systems in atest-strip assay is to obtain ahighly selective and sensitiveresponse while guaranteeingfast response times, that is,high delivery rates. Highly se-lective and sensitive immuno-logical responses are best ob-tained with high-affinity anti-bodies. However, such tightlybinding receptors are usuallycharacterized by a rather slowoff-rate or dissociation con-stant kd.

[13] Assuming acommon on-rate or associationconstant ka of 1 � 107

m�1 s�1 for

small-molecule hapten–high-af-finity-antibody interaction,[14, 15]

our TATP-affine sera with highaffinities[11] of K0�1 � 109

m�1

are characterized by kd�1 � 10�2 s�1, which can be consid-ered as moderate to slow off-rates.[13] The affinity of the an-tibody is approximately fourfold higher for hapten I, usedfor animal immunization, than for neat TATP,[11] so an alter-native hapten had to be developed. We thus opted for theslightly mismatching hapten II (Figure 1)[16] and synthesizedits reactive precursor III to be facilely anchored onto thesurface of the mesoporous material. In the next step, MSNswere added to a solution containing a high concentration ofsulforhodamine B to achieve efficient loading of the pores(solid S0) before III was anchored on the external surface toyield S1. For the preparation of the final gated sensing ma-terial S1-AB, S1 was suspended in phosphate-buffered saline(PBS) containing the antibody in PBS (pH 7.4) with 0.05 %bovine serum albumin (BSA) and sulforhodamine B. Opti-mization of the conditions was performed by checking arange of different serum dilutions. S1-AB was isolated bycentrifugation and washed with PBS. The solid was finallydried under vacuum overnight and stored at 4 8C. The pres-ence of BSA presumably helps in the capping of the poresby the antibodies, further reducing unspecific leaching ofthe dye from the sensor material. The S1-AB hybrids wereobtained as roughly spherical nanoparticles with a diameter

Figure 1. A) Schematic representation of the preparation of antibody-gated material S1-AB. B) Sensingscheme of analyte-mediated displacement of the stoppers and dye release. C) Chemical structures of TATPand the hapten derivatives I–III employed in this study.

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of approximately 100 nm and hapten III, dye, and antibodycontents of 0.17, 0.749, and 5.18 � 10�4 mmol (gSiO2

)�1, respec-tively; the complete details are given in the Supporting In-formation.

The response of S1-AB in the presence of TATP was stud-ied by suspending S1-AB (0.4 mg) in PBS (0.8 mL) atpH 7.4 (0.5 mg solid mL�1). This suspension was divided intotwo aliquots. To assess the effectiveness of pore closure, onefraction was mixed with PBS (2.375 mL) containing TATP(5 ppm) and the other fraction was only mixed with thesame amount of neat PBS. Fractions of both suspensions(0.25 mL) were then centrifuged for certain time intervals(0, 0.25, 0.5, 2, and 5 min) and the amount of released dyewas measured fluorometrically. The profiles of the deliverykinetics are shown in Figure 2 A. It is clear that the presence

of TATP induced a fast opening of the pores with subse-quent release of the entrapped dye, whereas virtually no re-lease was observed in the absence of TATP. The maximumdelivery of sulforhodamine B from S1-AB in the presence ofTATP (5 ppm) amounted to 25 % of the initial content ofdye in S1-AB. Following a similar procedure, dye deliveryfrom S1-AB was studied as a function of TATP concentra-tion (Figure 2 B), and the expected correlation was ob-served, in agreement with a displacement of the antibodyfrom the solid surface upon successful competition of TATPwith the binding sites (Figure 1). Analysis of the binding iso-

therms between the particle-bound antibody and TATP ac-cording to a Scatchard plot, often used to describe protein–ligand binding, revealed an apparent equilibrium constant ofK=4.5 � 105

m�1, the nonlinearity presumably arising from

the polyclonality of the sera. Regarding the limit of detec-tion (LOD) of the assay, TATP could be detected at concen-trations as low as 12.5 ppb (details of the analytical perform-ance are given in the Supporting Information). The amountof dye released in the working range of the assay at lowerTATP concentrations is about 70 times that of antibody dis-placed, clearly resembling signal amplification.

The selectivity of the capped material was studied bymonitoring the uncapping process in the presence of otherspecies, in particular of common explosives such as trinitro-toluene (TNT), hexogen (RDX), nitropenta (PETN), octo-gen (HMX), nitroguanidine (NG), and hexamethylene tri-peroxide diamine (HMTD); see Figure SI-1 in the Support-ing Information for chemical structures. Furthermore, thesynthetic precursors of TATP and III, namely, hydrogen per-oxide, acetone, and 7-oxooctanoic acid (7-oxo), as well asother synthetic cyclic peroxides, that is, tributanone triperox-ide (but-TP), tri-3-pentanone triperoxide (3-pent-TP), tri-2-pentanone triperoxide (2-pent-TP), diacetone diperoxide(DADP), and the structurally related, common crown ethercompounds 18-crown-6 (18C6) and 12-crown-4 (12C4), werealso tested. The uncapping ability of all these compounds ata concentration of 3 ppm is shown in Figure 3. Evidently,

from the structurally related compounds, only the analogous(yet in realistic scenarios not relevant) triperoxides but-TP(contains the same triperoxide ring as III), 2-pent-TP, and 3-pent-TP were able to partially induce dye release from S1-AB nanoparticles. Among the classical explosives onlyPETN and NG induced moderate dye delivery. Besides thelow detection limit for TATP, an important feature of thesystem is its excellent discrimination of TATP against H2O2,the cross-reactant most other methods suffer from.

Figure 2. A) Sulforhodamine B release measured by monitoring the fluo-rescence at 582 nm (lexc =564 nm) versus time for S1-AB in PBS(pH 7.4), in the absence (curve b) and the presence (curve a) of 5 ppmTATP. B) Sulforhodamine B release from S1-AB as a function of TATPconcentration in PBS (pH 7.4) after 5 min of reaction. The amount ofdye released was determined from the emission intensity at 582 nm (withlexc = 564 nm); error bars at 3s.

Figure 3. Relative release of sulforhodamine B from S1-AB in the pres-ence of 3 ppm of other common explosives and educts of TATP synthesisin PBS (pH 7.4); H2O2 is highlighted in gray. Inset: Relative release ofsulforhodamine B from S1-AB in the presence of 3 ppm of selected com-pounds that are structurally related to TATP. The amount of dye releasedwas determined fluorometrically (lem =582 nm, lexc =564 nm); error barsat 3s.

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COMMUNICATIONAssays Based on Antibody-Gated Dye-Delivery Systems

Encouraged by these results and after demonstrating thefeasibility of the combination of immunological indicationand mesoporous scaffoldings to design antibody-cappedmesoporous materials, we proceeded to integrate S1-ABwith a lateral-flow assay for the straightforward on-site de-tection of TATP. Such assays commonly rely on test stripsthat carry the (bio)chemical part of the detection systemand which, after dipping the strip into the sample solutionand evolution of the flow, develop a color that can be appre-ciated by eye. Few such assays are known for the determina-tion of small-molecule analytes of environmental (e.g., atra-zine),[17] diagnostic (e.g., morphine),[18] or terroristic (e.g.,saxitoxin)[19] concern at trace (lower ppb) levels; those thatare known commonly utilize antibody–gold nanoparticle(AuNP) conjugates or hapten–protein–AuNP conjugates,partly in combination with enhancer solutions. Alternatively,first reports on trace-level analysis by using quantum dot(QD) conjugates in strip-based assays and handheld fluores-cence readers instead of AuNP-based assays have been re-ported recently.[20] Inherent to both approaches are the factsthat such assays are rapid, sensitive, specific, cheap, andeasy to handle, clear advantages in routine applications byuntrained personnel or in emergency cases. However, bothAuNP- and QD-conjugate assays have a significant draw-back, that is, they require protein conjugates to travel theactive distance of the strip, which renders them prone toerrors due to unspecific binding. As can be deduced fromthe scheme sketched in Figure 1, our approach is different.The strip also contains an interaction and a detection zone(Figure 4). However, only the first zone A contains the(bio) ACHTUNGTRENNUNGchemistry—S1-AB (Figure 4 a)—that is necessary togenerate the response and the second zone B is an arbitraryarea at the solvent front in which a signal is collected (Fig-ure 4 c). If the investigated sample does not contain the ana-lyte (TATP) no dye release would be observed and nosignal would be detected in zone B. However, when TATP ispresent in the sample, uncapping takes place when the sol-

vent front reaches zone A because of competition betweenTATP and grafted III for the binding sites of the antibody,thus leading to formation of the more stable TATP–anti-body complex, and the liberated dye is transported at thesolvent front due to the flow conditions (Figure 4 b). Be-cause the scaffold MSNs have been chosen to be largeenough not to be transported by an aqueous flow in thestrips membrane, still capped and uncapped S1-AB areboth retained at the spot of deposition (Figure 4 c, zone A).Depending on the amount of TATP in the sample, the flowtransports a certain amount of released dye away fromzone A and a fluorescence signal can be detected at the sol-vent front in zone B.

A high-flow nitrocellulose membrane was selected as thesupport. Strips 0.5 �2.5 cm in size were prepared and S1-AB(0.5 mL) was deposited from suspensions of the sensing ma-terial (2 mg mL�1) on zone A with a micropipette. The stripswere then dipped into buffered solutions containing variousamounts of TATP. After 90 s of development, the test stripswere dried and the fluorescence was measured with a flowassay reader at 625 nm (lexc = 520 nm). The drying step is es-sential because varying amounts of residual liquid on thestrip influence the fluorescence of the dye. This step is alsothe time-limiting step, that is, when using a hair dryer (incold air blow mode), the assay can be completed in approxi-mately 2 min (90 s development plus 30 s drying), and whenletting the strip dry at room temperature under normal at-mosphere, approximately 8 min are required to obtain astable signal. When the PBS sample solution did not containTATP, a negligible fluorescence signal was recorded inzone B (Figure 5 A). However, when a similar experimentwas performed, for example, with a solution containingTATP (0.5 ppm), a clear signal was found in zone B (Fig-ure 5 B). To the best of our knowledge, this is the firstreport of a flow assay based on massive indicator releasefrom a nanoscopic chemical container device.

Following the same procedure as that described above,the effect of TATP concentration in the lateral-flow assayswas studied. The amount of dye released for each concentra-tion was calculated through the ratio between the area ofzone A and the total area of the test strip. An LOD of15 ppb of TATP was determined by using this simple proce-dure (Figure SI-2 in the Supporting Information). The effectof the educts of the synthesis of TATP, other explosives, andother peroxides was also assessed in cross-reactivity studiesand the results in terms of selectivity were similar to thoseobserved by using S1-AB in solution (Figure SI-3 in the Sup-porting Information). Moreover, besides the manual deposi-tion procedure described above, we also prepared strips forthe assay with an automated dispenser able to deliver drop-lets of �1 nL in volume from suspensions of S1-AB(5 mgmL�1) onto the strips; the final volume deposited was50 nL in these cases. The advantage of this procedure is thatan automated and reproducible preparation of the test stripsis possible (Figure SI-4 in the Supporting Information). Thesame selective uncapping process in the presence of TATPwas observed for these strips with detection limits of 40 ppb.

Figure 4. Design and principle of operation of the lateral-flow assay:a) S1-AB is deposited at zone A so that the strip can be convenientlydipped into the sample; b) the presence of the analyte leads to (partial)uncapping of the pores and release of the dye, which is transported at thesolvent front; c) after development and drying, zone B contains theamount of dye that corresponds to the amount of analyte in solution andzone A the residual, unreleased dye. Note that zone B does not containany specific focusing chemistry; the dye molecules are already sufficientlyfocused at the solvent front.

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These experiments have shown that the S1-AB hybrids arestable enough to be handled in an automated dispensingprocess by using a piezo-driven device. In contrast, due tosedimentation problems of the hybrid nanoparticles in thereservoir, even the use of high-end array spotters did notyield satisfactorily reproducible series of strips (Figure SI-5in the Supporting Information).

Finally, the strips from both deposition procedures,manual and dispenser, were tested under realistic conditionsand were able to detect TATP in tap water (pH 7.4) as wellas influent (pH 7.9) and effluent (pH 7.5) water of a sewagetreatment plant at lower ppb concentrations, when for in-stance PBS 10X solution (0.1 mL) was added to the sample(0.9 mL) prior to dipping of the strip. Regarding pH toler-ance, the system was tested in the pH range 1–12 andTATP-induced delivery was observed at any pH value. How-ever, below pH 4 and above pH 8.5, the delivery rates werereduced, presumably because at acidic pH, TATP starts todecompose[21] and at alkaline pH, the structure of the silicascaffold might be altered.[22] In addition, testing the assay on

samples with ethanol contents of 1–10 % v/v revealed thatenhanced release is observed only above 5 %, most likelydue to accelerated dissociation of the caps (for a detaileddescription and representative graphs, see Section 9 and Fig-ure SI-6 in the Supporting Information). Evidently, the strip-based assay is robust enough to accomplish the task of reli-ACHTUNGTRENNUNGably determining the presence of TATP in scenarios tolocate IED “bomb factories”, as described in the introduc-tion.

In summary, we have designed and prepared antibody-gated MSNs that are loaded with a rhodamine dye and thatcan be used for the determination of the presence of perox-ide-based explosive TATP with a lateral-flow fluorescencereader, thereby allowing for detection limits in the lowerppb range. The mechanism of the detection relies on a dis-placement of the antibody from the surface of the hybridmaterial because of highly affine antibody–TATP interac-tions, which release a much larger number of entrapped dyemolecules from the pores than antibodies are displaced. Thehigh selectivity of the antibody is retained in the gated ma-terial, thus allowing for a remarkable discrimination againstH2O2. System design and optimization led to straightforwardintegration into a lateral-flow assay without further treat-ment or conditioning of the test strips while guaranteeingfast overall assay times of �10 min. Moreover, besidesshowing a remarkable robustness, the inherent architectureof the assay relies on a single-step immunochemical reac-tion, that is, it can dispense with secondary (labeled) anti-bodies and enables ratiometric signal assessment in the pres-ence of the analyte by simple comparison of the fluores-cence signals in two zones. Because of the modularity of theapproach, the concept is easily generalizable and applicablefor many small-molecule analytes. For instance, assays canbe imagined that contain various hybrids of S1-AB(n) type,for which each material capped with a certain antibody isloaded with a different dye, thus envisioning multiplexedanalysis with such simple assays.

Acknowledgements

Financial support from the Spanish Government (MAT2009-14564-C04-01), the Generalitat Valenciana (PROMETEO/2009/016), the Innova-tionsfonds (BAM/Bundesministerium f�r Wirtschaft und Technologie),and BAMs PhD Program (M.A.W.) is gratefully acknowledged. E.C.thanks the Ministerio de Educaci�n for a fellowship. We are indebted toJ. Schenk, BAM-1.0, for help with the piezo dispenser and S. Fischer andW. Weigel, Scienion AG, Berlin, for support with the spotter.

Keywords: dyes/pigments · explosives · fluorescence ·immunoassays · mesoporous materials

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Figure 5. Signal obtained with the fluorescence reader (lexc =520 nm,lem =625 nm) in zones A and B after dye release and development of theflow assay in A) the absence and B) the presence of 0.5 ppm TATP inPBS buffer (pH 7.4); gray shades on the sketched strips resemble signalintensity.

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COMMUNICATIONAssays Based on Antibody-Gated Dye-Delivery Systems

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Published online: && &&, 0000

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Immunoassays

E. Climent, D. Grçninger, M. Hecht,M. A. Walter, R. Mart�nez-M�Çez,*M. G. Weller, F. Sancen�n, P. Amor�s,K. Rurack* . . . . . . . . . . . . . . . . . . . . &&&&—&&&&

Selective, Sensitive, and Rapid Anal-ysis with Lateral-Flow Assays Basedon Antibody-Gated Dye-DeliverySystems: The Example of TriacetoneTriperoxide

Set them free : Brightly fluorescentindicators that are loaded into meso-porous silica nanoparticle carriers,capped with bulky antibodies, arereleased into the lateral flow of a teststrip upon analyte arrival. Integrationof the system into a rapid, simple flowtest with fluorescence readout isapplied for the selective and sensitivedetermination of the presence of tri-ACHTUNGTRENNUNGacetone triperoxide (TATP) as a proto-type small-molecule analyte (seefigure).

Chem. Eur. J. 2013, 00, 0 – 0 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org

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COMMUNICATIONAssays Based on Antibody-Gated Dye-Delivery Systems