Methods 37 (2005) 65–72

www.elsevier.com/locate/ymeth

A “do-it-yourself ” array biosensor

Joel Golden a,¤, Lisa Shriver-Lake a, Kim Sapsford b, Frances Ligler a

a Naval Research Laboratory, Code 6900, 4555 Overlook Ave., SW, Washington, DC 20375-5348, USAb George Mason University, 10910 University Blvd, MS 4E3, Manassas, VA 20110, USA

Accepted 1 May 2005

Abstract

We have developed an array biosensor for the simultaneous detection of multiple targets in multiple samples within 15–30 min.The biosensor is based on a planar waveguide, a modiWed microscope slide, with a pattern of small (mm2) sensing regions. The wave-guide is illuminated by launching the emission of a 635 nm diode laser into the proximal end of the slide via a line generator. The eva-nescent Weld excites Xuorophores bound in the sensing region and the emitted Xuorescence is measured using a Peltier-cooled CCDcamera. Assays can be performed on the waveguide in multichannel Xow chambers and then interrogated using the detection systemdescribed here. This biosensor can detect many diVerent targets, including proteins, toxins, cells, virus, and explosives with detectionlimits rivaling those of the ELISA detection system.Published by Elsevier Inc.

Keywords: Biosensor; Evanescent wave; Array; Planar waveguide; Fluorescence

1. Introduction

The literature describing protein arrays or arrays ofligands for interrogating protein binding capability isextensive. Most of the existing technology uses methodsthat have evolved from the DNA microarray Weld,employing contact or non-contact arrayers for protein/ligand deposition and confocal scanners for reading theresults. There are signiWcant limitations aVecting the pro-gress in developing protein arrays, including problems inobtaining pure proteins as well as immobilizing themwithout compromising the functionality of the molecule;both of these problems are currently being addressed [1].In addition, the involvement of many smaller researchgroups in this Weld is limited by the cost of the equip-ment—particularly, high tech arrayers and confocal read-ers. In this paper, we describe a system for making protein

* Corresponding author. Fax: +1 202 767 9594.E-mail address: jgolden@cbmse.nrl.navy.mil (J. Golden).

1046-2023/$ - see front matter. Published by Elsevier Inc.doi:10.1016/j.ymeth.2005.05.010

or ligand-based arrays and a simple, low cost reader thatcan be assembled with a minimum of eVort.

Capture antibodies (or other binding molecules) aredeposited on the microscope slide to fabricate the arraysusing a very simple technique [2,3]. First, NeutrAvidin iscovalently attached to the cleaned slides [4]. Then apolydimethoxysilane (PDMS) block with molded parallelchannels is clamped to the slide using a chuck milled outof Plexiglas. The protein or ligand to be immobilized,modiWed with the attachment of a biotin group, is insertedinto each of the channels for at least an hour. Afterremoval of any unbound protein, rinsing, and blocking,the waveguide is ready for use. For assays, a similarPDMS block with the channels running perpendicular tothose in the Wrst block is attached to the slide, and samplesand Xuorescent reagents are Xowed through these chan-nels. The exact protocol can be varied, depending on theapplication of interest. After the assay, the waveguide isrinsed, dried, and placed in the reader for analysis.

The reader consists of a Peltier-cooled CCD camera, a635nm diode laser equipped with a line generator, emis-sion Wlters, and a scaVold for holding the microscope

66 J. Golden et al. / Methods 37 (2005) 65–72

slide-waveguide in place. The image collected by the CCDis transmitted to a computer (PC or laptop) via a Wrewireconnection, and data analysis is performed in a semi-auto-mated manner to accommodate variable numbers ofspots. This system has been used to measure binding con-stants for antibody–antigen and sugar–toxin interactions[5]; to perform all four diVerent types of immunoassays[6]; to detect pathogens and toxins in complex samples [7].Because multiple samples can be interrogated using multi-ple proteins and/or ligands simultaneously, this system isparticularly useful for characterization of speciWcity andavidity. Fig. 1 shows an example of multianalyte assays.

The system described here is simple to fabricate and easyto use. It has been used extensively by undergraduates andeven high school students working as summer interns. Sen-sitivities equivalent to ELISAs are routinely obtained in15min assays [8]; higher sensitivities can be obtained byincreasing the incubation times [9]. Furthermore, a fullyautomated version has been recently tested for Weld opera-tion [10]. The purpose of this paper is to provide instruc-tions for assembling the array reader for the analysis ofarray-based assays performed oZine and for fabricating thearrays on avidin-coated slides using the PDMS channels.

2. Materials and methods

2.1. Planar waveguide assays

In these planar waveguide assays, the evanescentwave interacts with and excites Xuorophores at the sur-

face of the waveguide, and the resulting Xuorescence ismeasured using a detector [11–17]. Assays can be per-formed on the patterned microscope slides with theintroduction of Xuids using PDMS Xow chambersaccessed via syringe needles. After the assay, the PDMSXow chambers are removed and the slide is placed on thesensor for interrogation. The slides demonstrate little, ifany, loss of signal over periods of weeks to months, solong as the slides are rinsed before storage and kept in adark, cool place.

2.2. Assembly of array reader

2.2.1. System designA schematic of our desktop array biosensor is shown

in Fig. 2. Components are mounted onto a vertical opti-cal breadboard with posts, post holders, and translationstages (Newport Corporation). An advantage of thisdesign is that the imaging path (detection) is perpendicu-lar to the excitation light path, which limits the amountof excitation light reaching the imager to only the lightthat is scattered by the waveguide. This design alsoallows another laser and Wlter set to be substituted forthe diode laser to perform assays with dyes that excite atother wavelengths.

A cardboard tube painted Xat black on the inside canbe placed over the lens (17 mm C-mount lens, SchneiderOptics, Hauppauge, NY) to reduce stray light. Fordetecting CY5 or AlexaFluor 647, we found the combi-nation of a 700 § 35 nm bandpass Wlter (P70-700-S,Corion, Franklin, MA) and a 665 nm longpass Wlter

Fig. 1. Image of a slide showing sandwich assays for Clostridium botulinum toxoid A (Bot. Tox. A), Staphylococcal enterotoxin B (SEB), and Cam-pylobacter jejuni (Strain ATCC35918; Camp. J22). The right and leftmost columns are positive control assays (chicken IgG). Sample was introducedinto each (horizontal) channel with combined concentrations of Bot. Tox. A, SEB, and Camp J22 as shown on right side of the Wgure. Then a solu-tion of a mixture of CY5-labeled antibodies for each analyte was introduced to the channels, along with CY5-labeled chicken IgG.

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J. Golden et al. / Methods 37 (2005) 65–72 67

(RG665, Schott Glass, Duryea, PA) to be useful. Theemission Wlters can be placed above the cardboard tubeto make them easy to replace; an adjustment in camerafocus will likely be needed after changing emissionWlters. The light path between the camera lens and theslide needs to be carefully shielded from stray light.Black photographic tape is ideal for this purpose.

The laser beam passes through a line generator(Lasermax) and is then launched into the end of theslide. The expanded beam should be carefully alignedwith the slide edge so that the excitation light evenly illu-minates the surface. The excitation light is launched intothe slide at an angle of approximately 30° to increase thepower in the evanescent Weld while maintaining totalinternal reXection.

For the slide mount, a 1 mm deep recess was milledinto aluminum stock to Wt the slide (Fig. 3). The perime-ter of this region was raised about 1/2 mm to keep the

Fig. 2. Schematic of desktop biosensor design.

Fig. 3. Slide mount. Four inch square aluminum has a 1 mm recessmilled in which to mount the slide. Hatched area is slightly raised tokeep slide surface from touching the aluminum.

surface of the slide from touching the aluminum(to avoid light scattering). A 1-in.2 hole was made inthe mount near the distal end of the slide for viewingthe patterned region. This mount provides reproduciblepositioning of the slide such that minimal alignmentof the excitation beam is required for each slide. The alu-minum was anodized or blackened to reduce lightscattering.

2.2.2. ImagerThe imager we have used in this system is a Peltier-

cooled CCD camera (Retiga 1300, Q-imaging) with a2/3 in. CCD and 12 bit digitization. A major advantageof this camera is that it has a Wrewire connection fortransferring images, eliminating the need for a framegrabber. A laptop or PC captures the images through theWrewire connection [10]. High sensitivity is not a require-ment for the camera, although sensitivity should be suY-

cient for low light measurements. In addition, stabilityand suYcient dynamic range is desirable. In a directcomparison of a CCD, CMOS imager and a diode array,the CCD was still the imager of choice [18]. Our earlyexperiments were with room temperature CCDs, whichproved less satisfactory due to greater Xuctuations in sig-nal to noise, likely due to temperature changes. The Pel-tier-cooled cameras proved to have a much more stablebaseline. It is not necessary to use an expensive imagingsystem with liquid nitrogen cooling, since there is alwaysa non-zero background due to scattered light and Wlterbleedthrough.

2.3. Image collection and analysis

After performing the assays described below, the dryslide is placed on the slide mount with the assay side fac-ing up (away from the camera). The expanded laserbeam is aligned with the proximal edge of the slide toensure even illumination. Camera exposure time isadjusted to obtain an image of proper contrast. After theimage with Xuorescent spots is captured, it needs to beanalyzed to determine signal levels for each of the assaysperformed. A program written in LabWindows(National Instruments) imports the image and reportsaverage pixel values for the Xuorescent spots (Fig. 4).The region surrounding the spot (but still within the Xowchannel) reXects the level of non-speciWc binding and isconsidered background to which the signal in the assayspot must be compared. Signal from positive and nega-tive control channels can also be used to validate signalobtained from the assay spots.

2.4. Preparation of PDMS molds and chambers

Plexiglas plates (»1/2 in. thick) are milled with anumerically controlled milling machine (Techno Isel) toform the molds for the patterning and assay PDMS Xow

68 J. Golden et al. / Methods 37 (2005) 65–72

chambers (Fig. 5). Fins approximately 1 mm wide andtall are milled in the Plexiglas to form the channels in thePDMS through which the solutions will Xow.

The PDMS Xow chamber gaskets (patterning andassay formats) are formed by mixing NuSil MED-4011(Nusil Silicone Technology, Carpintera, CA) Part A andPart B at a 10:1 w/w ratio in a large plastic disposablebeaker. The mixture is degassed in a vacuum oven atroom temperature (RT) until all the bubbles areremoved (»30 min). The PDMS is poured into the Plexi-glas mold. This mold is placed in the vacuum for further

Fig. 4. Screen capture of slide image in data analysis program writtenin Labwindows. Mean pixel value and standard deviation are calcu-lated for each assay spot. The regions adjacent to each assay spot arethe background regions.

Fig. 5. Mold for making PDMS Xow chambers.

degassing until all the bubbles are at the surface, becausebubbles near the Wns can cause problems in the WnalPDMS Xow chamber. After degassing, the mold is left atRT to cure, or it may be placed in a 60 °C oven for30 min. Some caution is advised with the oven method,as the Plexiglas mold can warp at higher temperatures.After curing, the PDMS is removed from the mold,trimmed, and washed with soap to remove any residue.For many applications, it is also useful to incubate thePDMS in 1% bovine serum albumin (BSA) prior to useto prevent non-speciWc adsorption of molecules to thewalls of the channel.

2.5. Patterning and assay chucks

The PDMS Xow channel gasket is placed on top ofthe slide such that the antibodies are immobilized in pat-terns over the distal 1/3 of the waveguide. (The proximal2/3 of the waveguide provides mode-mixing of the light,producing even illumination over the detection area).This region has to line up with the hole in the slideholder in the detection system. To apply pressure evenlyon the PDMS gasket, we designed a chuck made of twoPlexiglas plates (Fig. 6). One plate has a recess to Wt theslide and six tapped holes around the perimeter. Theother plate is the same size and has holes that line upwith the tapped holes. This plate also has access holes forthe syringe needles used to pierce the PDMS at bothends of the Xow channels for Xowing the reagents. Forperforming assays, similar chucks have been prepared toclamp the assay PDMS chambers.

2.6. Preparation of NeutrAvidin-coated slides

2.6.1. MaterialsPotassium hydroxide (KOH) and phosphate buVered

saline packets, pH 7.4 (PBS, Sigma P-3813) were pur-chased from Sigma Chemical. Mercaptopropyltriethoxysilane (MTS) and N-[�-maleimidobutyryloxy]succini-mide ester (GMBS) were obtained from Fluka Chemical.Toluene and methanol were purchased from AldrichChemical. NeutrAvidin was obtained from Pierce Chem-ical. The microscope slides were purchased from A.J.Daigger, and the glove bag was obtained from IRS(Institute for Research in Industry).

1. Microscope slides are numbered by etching with adiamond scribe in the lower right hand corner. Twoslides are placed back to back into a Coplin jar withthe etched numbers to the outside.

2. A potassium hydroxide cleaning solution is preparedby adding 10 g KOH to 100 ml methanol. This solu-tion is poured over the slides to completely immersethem. The slides are exposed to the cleaning solutionfor 30–60 min. The slides are exhaustively rinsed with18 m� water until no schlieren lines are visible there.

J. Golden et al. / Methods 37 (2005) 65–72 69

The slides are then dried under a nitrogen stream. Theslides should visually appear to be spotless. Thecleaned slides are then placed in a new clean, dryCoplin jar. Safety note: the cleaning solution isextremely basic. Proper protective equipment shouldbe worn.

3. The next step is performed in a glove bag under nitro-gen. One milliliter MTS is added to 50 ml toluene andthoroughly mixed. The silane solution is added to theCoplin jar containing the cleaned slides. The slides areincubated with the silane solution for 1 h.

4. The slides are rinsed three times in toluene, then driedwith nitrogen, and placed in a new dry Coplin jar. Theslides should still appear pristine.

5. A 1 mM GMBS solution is prepared by dissolving12.5 mg GMBS in 250�l DMSO and brought up tovolume (43 ml) with ethanol. The slides are incubatedin this solution for 30 min. Time is important at thisstep as the reactive groups on the GMBS degradeover time in this solution. After 30 min, the slides arerinsed with 18 m� water extensively and placed in anew Coplin jar.

6. A 40 mM NeutrAvidin solution in PBS is added tothe slides. The slides are incubated overnight at 4 °C.The next day, the slides are rinsed three times withPBS. They are stored in PBS at 4 °C until it is time forpatterning.

2.7. Patterning

2.7.1. MaterialsThe buVers used in this step include the patterning

buVer (10 mM phosphate buVer, 10 mM sodium chlo-ride, and 0.05% Tween 20, pH 7.4) and PBSTB (phos-

phate buVered saline, 0.1% Tween 20, and 1 mg/mlbovine serum albumin, pH 7.4). The patterning PDMSgaskets and chucks described above are used in this pro-cedure along with 1 ml syringes with 25 gauge needles.The recognition molecules that are to be patterned needto be biotinylated. There is some variation on thismethod, depending on the recognition molecule [19].

1. A NeutrAvidin slide is rinsed in 18 m� water anddried with nitrogen. It is placed on the bottom of thepatterning chuck with the number side up. Next, thePDMS patterning gasket is placed over the slide atthe distal end (away from number) with channelsbeing formed by the slide and the PDMS. Finally, thetop plate of the patterning chuck is placed over thePDMS and four screws are inserted and tightenedevenly. The screws should be Wnger tight; over-tight-ening will result in slide breakage; too loose and thechannels leak into each other. Open syringes with nee-dles are placed in each channel as an eZuent reser-voir.

2. Fig. 7 shows the assembly ready for patterning. A60–100 �l solution containing the recognition mole-cule (biotinylated antibody for sandwich assays andbiotinylated analyte for competitive assays) in thepatterning buVer is injected into the appropriatechannel. In our sandwich immunoassays, 10�g/mlbiotinylated antibodies are routinely used for pattern-ing. In the competitive immunoassays, the biotinyla-ted analyte concentration varied from 0.2 to 5�g/ml.For multianalyte sensing, diVerent biotinylated recog-nition molecules can be immobilized in each of thepatterning channels. For many of our studies, a posi-tive control using an irrelevant analyte was performed

Fig. 6. Patterning chuck. Slide is inserted into the recess. PDMS is placed on top of the slide and the top plate is tightened with four screws to make awatertight seal.

70 J. Golden et al. / Methods 37 (2005) 65–72

in the outermost patterning channels and a negativecontrol was injected into one of the middle channels.Antibodies for the positive control (usually anti-chicken IgY) were immobilized in the control chan-nels, and for the negative control, buVer was Xowedthrough a patterning channel. The biotinylated recog-nition molecule is allowed to incubate with the Neu-trAvidin-coated slide overnight at 4 °C.

3. Each channel is rinsed with 1 ml PBSTB followed byair. The patterned slide is removed from the pattern-ing PDMS and chuck. The slide is put into a 50 mlcentrifuge tube containing 10 mg/ml bovine serumalbumin in phosphate buVer, pH 7.4, for 20 min toblock the slide. The slide is then rinsed with 18 m�water and dried with nitrogen. The dried patternedslide is stored at 4 °C until use.

3. Assays

We have developed assays for over 40 diVerent ana-lytes using the array biosensor. The majority of theassays are for large molecules, such as bacteria and pro-tein toxins [3,20]. Large molecules lend themselves tosandwich immunoassays as there is more than one epi-tope (binding site) on the molecule. Most of the assays(sandwich and competitive) have detection limits in thelow ng/ml range with the exception of ovalbumin, which

Fig. 7. Photo of slide in the patterning assembly ready for patterning.Syringe needles are carefully inserted through the PDMS into eachXow channel. Solutions containing capture antibodies are in the closedsyringes on the left side. Syringes on right side are inserted to allowXow through the chambers and are used as eZuent reservoirs.

presently has a detection limit of 25 pg/ml [23]. Theseassays have also been tested in a variety of sample matri-ces. Bacteria or toxins were spiked into clinical (blood,urine, and nasal swabs) [2], environmental [21], and food(juices, meat extracts, and pasta extracts) samples [7,22–24]. In some cases, there was a slight loss of sensitivitybut most of the assays have the same detection limits inthese diverse matrices. If the pH of the matrix is too low,it needs to be adjusted prior to analysis, such as forcoVee or tomato juice.

3.1. Sandwich immunoassay

3.1.1. MaterialsIn addition to the test or spiked sample, a 5–10 �g/ml

Xuorescently labeled tracer antibody is used. For the635 nm laser and Wlter set described here, the Xuoro-phore can be either Cy5 (Amersham Biosciences) orAlexaFluor 647 (Molecular Probes). In this step, theassay PDMS gasket and chucks that were described ear-lier are employed as well as the PBSTB buVer and syrin-ges. To perform the assay, a multichannel pump is usedto draw the solutions over the slide (SARAH, BarnstadtCo.).

The patterned slide is placed into the bottom of theassay chuck. The assay PDMS gasket is laid over theslide such that the channels run perpendicular to thoseof the patterning PDMS. The top of the assay chuck isattached in the same fashion as the patterning chuck.Screws are tightened to clamp the PDMS onto the slide.

In sandwich immunoassays, the analyte of interest issandwiched in between the capture antibody (the immo-bilized antibody) and the tracer antibody (antibody insolution labeled with Xuorophore). In our studies, thisassay was performed as a “two-step” assay (sample fol-lowed by tracer).

1. The assay chamber is attached to the pump by insert-ing 25 gauge needles through the PDMS into the endof each channel. Each needle is attached to a separatechannel on the pump.

2. Open syringes (1 ml) with 25 gauge needles areinserted into the PDMS at the other end of the chan-nel. Reagents to be Xowed through the channel areplaced in the syringe reservoirs.

3. The channels are Wrst rinsed with 1 ml PBSTB at aXow rate of 1 ml/min to make sure the system is work-ing properly (Xowing without leaking).

4. Next, 0.8 ml of the aqueous sample is Xowed over theslide at the rate of 0.1 ml/min. In some assays, 8 mindoes not provide enough interaction time to get opti-mal sensitivity. In those cases, the tubes leading fromthe pump are placed back into the syringes to create arecycling system. After 15 min, the recycling isstopped and the test sample removed from the chan-nels by increasing the pump speed to 1 ml/min. In

J. Golden et al. / Methods 37 (2005) 65–72 71

cases of large particulates in aqueous samples, theparticulates are removed by centrifugation and thesupernatant is used for the assay.

5. The channels are then Xushed with 1 ml PBSTB Xow-ing at 1 ml/min.

6. The next step is the tracer solution. At a Xow rate of0.1 ml/min, 0.4 ml of the tracer solution is Xowedthrough the channel. In most of our assays, the Xuo-rescently labeled antibodies are used at a concentra-tion of 10 �g/ml. The antibodies were labeled witheither CY5 or AlexaFluor 647.

7. After the tracer solution, the channels are Xushed alast time with 1 ml PBSTB followed by air.

8. The slides are removed from the assay chuck, rinsedwith 18 m� water, and dried with nitrogen. The slideis then imaged as described in Section 2.3.

3.2. Competitive immunoassay

For smaller molecules, binding sites are limited, socompetitive immunoassays are employed. Our recentfocus for the competitive assays has been on mycotoxindetection [25]. An important factor with competitiveimmunoassays is careful selection of the biotinylatedanalyte. The antibody should have similar aYnities forthe free and immobilized analytes. A word of caution,the linker length (between biotin and analyte) aVects therelative aYnity of the antibodies.

3.2.1. MaterialsThe test sample contains 90% sample and 10% 10£

PBSTB containing a known concentration of Xuores-cently labeled antibodies. As with the sandwich assay,the same assay PDMS gaskets, Plexiglas chucks, andpump are employed. The patterned slide is attached tothe assay gaskets and chucks as in the sandwich immu-noassay.

In the competitive immunoassay, the immobilizedbiotinylated analyte competes with the free analyte inthe sample for binding to the Xuorescently labeled anti-body in solution. Unlike the sandwich assay where thereis an increase in Xuorescence corresponding to the con-centration of the analyte in the test sample, a decrease inXuorescence is observed as the analyte concentrationincreases. This assay is performed as a “single-step”assay (sample and tracer together). The slides wereattached to the pump as described with the sandwichimmunoassay.

1. First, 1 ml PBSTB is Xushed through the channels at1 ml/min to verify Xow and check for leaks.

2. Next, the test sample containing the Xuorescentlylabeled antibody (0.8 ml) is passed through the chan-nels at a Xow rate of 0.1 ml/min.

3. The last step is Xushing the channels with 1 ml PBSTBat 1 ml/min and removing all liquid.

4. The slide is removed from the assay chuck, rinsedwith 18 m� water, and dried with nitrogen. The slideis ready for imaging as described in Section 2.3.

4. Conclusions

The assays described here are performed oZine, butwe have developed a portable version of this array bio-sensor that performs multiple assays in multiple samplessimultaneously using automated Xuidics [10]. Evanescentwave Xuorescence measurements are particularly wellsuited for detection in real-world samples because theanalyses can be performed with minimal matrix eVects[2]. However, automated assays require the use of a Xowchamber attached to the waveguide. Any chamber thattouches the surface of the slide will absorb and scatterthe excitation light. To circumvent loss and light scatter-ing, we prepared slides with a reXective coating wherethe chamber contacts the surface, allowing the wave-guide to be used with a Xow chamber [10].

The array biosensor described here can be used withthe modiWed slides and Xow chambers to measure bind-ing of recognition molecules to analytes in real time[5,26]. From such measurements, the kinetics of the bind-ing event can be determined.

The array biosensor combines techniques in the Weldsof biochemistry, immunology, electronics, and optics toyield a sensitive detection system capable of detecting awide variety of analytes. The kinds of analytes that can bedetected are only limited by the availability of appropri-ate antibodies or other capture ligands. This biosensor isalso adaptable to detect a variety of Xuorophores by sim-ply changing the Wlters and laser excitation wavelength.

Acknowledgments

The work was supported by NIH Grant EB02002 andthe Department of Defense. The views expressed hereare those of the authors and do not represent those ofthe US navy, the US Department of Defense or the USGovernment.

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