Highly sensitive phage-based biosensor for the detection of β-galactosidase

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ARTICLE IN PRESS+ModelCA-228277; No. of Pages 7

Analytica Chimica Acta xxx (2007) xxx–xxx

Highly sensitive phage-based biosensor for thedetection of �-galactosidase

Viswaprakash Nanduri a, Shankar Balasubramanian b, Srinivas Sista b,Vitaly J. Vodyanoy a, Aleksandr L. Simonian b,∗

a Department of Anatomy, Physiology and Pharmacology, Auburn University, Auburn, AL, United Statesb Department of Materials Engineering, Auburn University, Auburn, AL, United States

Received 21 October 2006; received in revised form 20 February 2007; accepted 26 February 2007

bstract

Development of real-time sensor based on the target-specific probe that make possible sensitive, rapid and selective detection and monitoring ofhe particular antigen molecules could be of substantial importance to the many applications. Because of its high specificity to the target molecules,xcellent temperature stability, and easy production, bacterial phage might serve as a powerful biorecognition probe in biosensor applications.ere, we report extremely sensitive and specific label-free direct detection of model antigen, �-galactosidase (�-gal), based on surface plasmon

esonance (SPR) spectroscopy. The �-gal specific landscape phage 1G40 has been immobilized on the gold surface of SPR SPREETATM sensorhip through physical adsorption [V. Nanduri, A.M. Samoylova, V.Petrenko, V. Vodyanoy and A.L.Simonian, Comparison of optical and acousticave phage biosensors, 206th Meeting of The Electrochemical Society, Honolulu, Hawaii, October 3–8, (2004)]. Another non-specific to the-gal phage, a wild-type phage F8-5, was used in the reference channel. The concentration-dependent binding of �-gal in both channels weressessed by monitoring the sensor optical response as a function of time under different experimental conditions, and the concentration of �-gal wasomputed in differential mode. Concentrations of �-gal between 10−12 M and 10−7 M could be readily detected, with linear part of calibration curveetween 10−9 M and 10−6 M. When �-gal was pre-incubated with different concentrations of free 1G40 phage prior to exposure to the biosensor,oncentration-dependent inhibition was observed, indicating on biosensor high specificity toward �-gal. Apart from a flow through mode used toeliver the samples to the surface for the SPR sensor, batch mode sensing was also employed to study the binding of �-gal to immobilized phage

n the SPR sensor surface. Experiments using a flow through mode provided more consistent results in the full dose range and showed higherensitivity as opposed to the batch mode studies. The mean Kd and binding valences for the flow through mode studies was 1.3± 0.001 nM and.5± 0.03, in comparison to 26± 0.003 nM and 2.4± 0.01 for the batch mode studies. The average thickness of phage 1G40 adlayer depositedhrough flow through and batch mode was 3± 0.002 and 0.66± 0.001 nm, respectively.

2007 Elsevier B.V. All rights reserved.

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eywords: Surface plasmon resonance; Sensor; �-Galactosidase; Filamentous

. Introduction

Biosensors are nowadays used in a wide range of fields fromhe detection of pollutants in the environment to the detec-ion of pathogens in the food industry [3]. Whatever function

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biosensor may perform, it is essential to use an appropriateiorecognition probe so as to achieve a high degree of sensitiv-ty and specificity to the target it is designed for. The purpose

∗ Corresponding author at: 275 Wilmore, Auburn, AL 36849, United States.el.: +1 334 844 4485; fax: +1 334 844 3400.

E-mail address: [email protected] (A.L. Simonian).

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003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2007.02.071

iophage; Biosensor

f this study was to investigate a model protein–probe specificnteraction providing a background for sensitive detection of a

odel protein. Escherichia coli �-gal, which involves in the fer-entation of lactose and is a well known marker for the detection

f coliforms was selected for these studies as a typical modelrotein, and filamentous phage specific to �-gal was used ashe model probe [4,5]. The binding of �-gal in solution to phagemmobilized on gold surfaces has been reported earlier [6]. Otherignificant applications of this enzyme include being used as a

e-based biosensor for the detection of �-galactosidase, Anal. Chim.

arker for gene cloning and construction of plasmids [7] ands fusion partner protein [8]. The four identical subunits [9] ofhis 116,000 Da protein is especially useful in studying multi-alent interactions. SPREETATM, a surface plasmon resonance

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deliver all solutions to the gold surface (Fig. 1a), a batch modesetup (Fig. 1b) was also designed and tested. A Teflon blockwas milled to smoothness using a Microlux milling machineto dimensions of 26.13 mm× 24.95 mm with a thickness of

Fig. 1. (a) The flow through mode setup consisting of source point and a wastecollector for solutions flowing in and out of the SPR system, a peristaltic pump

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SPR) sensor was used as the sensing platform. Through thislatform, the sample to be investigated can be studied withoutny labeling, in continuous real time mode. Recent years haveeen the use of SPR for the detection of E. coli enterotoxin [10];creening of compounds interacting with HIV-1 proteinase [11];nalysis of biomolecular interactions with biomaterials [12];uantification of human IgE [13]; structural analysis of humanndothelin-1 [14] and characterization of thin film assembly15,16]. SPREETATM, described in details earlier [17,18] haseen successfully employed for the detection of mutant DNA19,20]; immobilization of DNA probes [21,22] and detectionf Hg2+ [23]. The compactness of the SPREETATM provides aistinct advantage over other platforms for deployment in theeld. The fact that the SPR sensor surface can be reused bymploying simple regeneration reagents and techniques adds ono the advantages of the SPR platform over other contemporaryiosensor platforms in use today [16].

. Materials and methods

.1. Phage

The filamentous bacteriophage 1G40, used as a probe in thistudy is a virus that possesses a single stranded DNA encap-ulated in a protein sheath. It was selected from a library oflamentous phages as described [4,24] and are flexible rod-liketructures about 1.3 �m in length and about 10 nm in diameter.he protein sheath is made up of both major and minor coatroteins. The target for designing the probe is the major coatrotein, pVIII, of which there are several thousand copies. Aild-type phage, F8-5, was used as a reference probe without-Gal specificity. The total number of viral particles present inhage preparations was determined by spectrophotometer usinghe formula [25]:

irions(vir)/mL = (A269 × 6× 1016)

number of nucleotides in the phage genome

here A269 is absorbance at 269 nm. For the recombinant phagessed in this work (9198 nucleotides), the formula:

Absorbance unit, (AU)269 = 6.5× 1012 vir mL−1 was used toetermine the concentration of phage particles in a solution [2].

.2. β-Galactosidase

Escherichia coli �-galactosidase was obtained from Sigmahemical Co. (G5635) as a lyophilized powder and was dis-

olved in Dulbecco’s phosphate buffered saline (DPBS) at finaloncentration of 2.4 mg mL−1.

.3. Solutions, reagents and tubing

Dulbecco’s phosphate buffered saline solution [DPBS] wasbtained from BioWhittaker Inc., (17-512F). Tris-buffered

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aline (TBS) was prepared from Tris crystallized free base;isher Scientific, BP 152-1; TBS-Tween [TBS containing 0.5%v/v) Tween]. Bovine serum albumin (BSA) Fraction V; Sigmahemical Co. A2153; 50 mg mL−1 stock dissolved in Millipore

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PRESSica Acta xxx (2007) xxx–xxx

ater was filter-sterilized and stored at 4 ◦C. 0.64 mm (inneriameter) silicone tubing (Cole Parmer, cat #: 07625-22), 3 mLatex syringes (Becton and Dickinson) and 2 mL polypropy-ene cryogenic, round bottomed tubes cylinder (Corning, cat:EW-44351-15).

.4. Miniature two-channel SPR sensor

SPREETATM, a dual channel miniature (4 cm× 1.3 cm×.8 cm) sensor (Texas instruments) belongs to the class ofPR sensors that use angle interrogation was used as a detec-

ion platform. The various components of the device and theeasurement techniques are as described [1,26,29–31]. Theavelength of the light employed for interrogation of the angle

hange is 840 nm. It should be mentioned that all the experimentsere conducted at room temperature, which did not fluctuate

ignificantly at any point during the duration of study.

.5. SPR sensor batch mode setup

Apart from the flow through mode setup that was employed to

e-based biosensor for the detection of �-galactosidase, Anal. Chim.

nd personal computer (PC) for collecting data and offline analysis. (b) Theomponents of the batch mode setup chiefly consisting of a teflon block with aeservoir for holding the solutions. A tight seal of the Teflon block against thePR sensor surface was ensured by the use of a rubber gasket. All figures not tocale.

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1.16 mm. Four holes were drilled in the corners of the Teflonlock to match those on the front edges of the black anodizedide plates that were used to sandwich the SPEEETATM sen-or in between them. The screws were used to hold the Teflonlock to the sensor/side plates, and the whole setup was clamped.

reservoir of 9.62 mm× 4.95 mm× 11.16 mm, the bottom ofhich aligned with the gold surface of the sensor also was cut

nto the Teflon block and formed a measurement cell. A rubberasket of similar dimensions was shaped and applied betweenhe Teflon block and the sensor to provide a tight seal. A smallnlet of 2.95 mm (inner diameter) was drilled near the bottom ofne side of the reservoir to facilitate delivery of different solu-ions in a plane parallel to the gold surface of the sensor. Thisype of delivery was found to be most effective for stability ofhe sensing (phage) layer. A latex syringe of 3 mL capacity (Bec-on and Dickinson) was used to deliver the solutions. Separateyringes were employed for the delivery of different solutions.he whole setup was then housed in a black box so as to prevent

nterference from background light.

. SPR sensor preparations

In all experiments SPREETATM, a scaled down, highly inte-rated surface plasmon resonance (SPR) sensor was used. AllPR sensors were plasma cleaned in Argon using PlasmodM system (Manchester Inc.) at 1 T for 5 min prior to beingmployed in any tests. Immobilization of the phage to the goldurfaces was through physical adsorption for both flow throughnd batch mode of investigations [1]. In the flow through mode,oth the inlet and outlet silicone tubing were of uniform internaliameter (0.64 mm). Both inflow and collection solution tubesere round bottomed, polypropylene cryogenic tubes of uniformiameter [Corning]. Both of the above conditions were consis-ently maintained so as to ensure absence of any air bubbles inhe system.

.1. β-Galactosidase binding measurements

The photodiode array response changes as a measure ofefractive indices/units were recorded and transferred to a per-onal computer via an RS 232 interface card. All the data werebserved in real-time using the “Multispr” (version 10.68) soft-are which also enables to store the data that can be analyzedffline. The software also provides such information of the sig-al as either refractive index or refractive units versus time;hickness and coverage of adlayer (adsorbate) and angle.

.1.1. Kinetics of bindingBased on the basic thermodynamic principle governing

hage–�-gal interactions in solution can be expressed by:

h+ �gka←→Ph�g (1.1)

Please cite this article in press as: V. Nanduri et al., Highly sensitive phagActa (2007), doi:10.1016/j.aca.2007.02.071

kd

here, Ph represents free phage, and �g represents free �-al, Ph�g is the phage–�-gal complex, and ka and kd are thessociation and dissociation rate constants, respectively. The

effc

PRESSica Acta xxx (2007) xxx–xxx 3

quilibrium constant, or the affinity, is given by:

= ka

kd= [Ph�g]

[Ph][�g](1.2)

hile both the association and dissociation rates are relativelyuicker in solution, the association of the phage–� gal complexs mostly affected by the diffusion of the reactants, whereashe dissociation is mainly determined by the phage affinityo the �-galactosidase and the strength of the phage–�-galond. Whatever maybe the immobilization technique employed,mmobilization can alter the properties of the phage or �-gal,hus affecting the binding kinetics [2].

The Hill plot is derived by plotting log θ versus log [�g] where�g] is the ligand concentration and θ is given by the equation:

= Y

1− Y(1.3)

here Y = �S/�Smax [2] and S denotes the signal obtained (aftereducting the baseline).

The Hill plot also enables us to derive the dissociation con-tant Kd, and the binding valency which indicates the numberf sites available for binding. Lower Kd values denote strongerinding and higher sensitivity of the sensor. All binding wasetermined and quantified using the Hill plot, [27] and all resultsuch as Hill coefficient and effective dissociation constant Kdnd the binding valences were determined as described [2].

.1.2. Flow through modeIn the flow through mode, a cleaned, gold surface of the

ensor was exposed to 250 �L of recycling phage suspen-ion (3.2× 1011 vir mL−1) at a low, recycling flow rate of50 �L min−1 till saturation was achieved (approximately 3 h)nd followed by washing with Dulbecco’s phosphate bufferedaline (DPBS). The low flow rate was opted, as preliminary trialssing higher flow rates (data not shown) resulted in an unstablehage adlayer. Bovine serum albumin (BSA) 2 mg mL−1 wassed to block the sensor surface. The sensor was then exposed toraded concentrations of �-gal solutions (0.0032–210 nM) withntermediate washes of DPBS, and the changes in the refractivenits were recorded as described [1]. The flow through modead the added advantage of a dual channel, so that while onehannel served as the working, the second one was used as aeference The flow through setup (Fig. 1a) essentially consistsf two source points from which the solutions for the two sep-rated channels are pumped using a peristaltic pump (Regloigital, Ismatec SA, Switzerland) onto the gold surface of thePR sensor surfaces; a waste collector and personal computer

hat collects the data through an RS 232 interface for offlinenalysis.

.1.3. Batch modeIn the batch mode method, the cleaned SPR sensors were

e-based biosensor for the detection of �-galactosidase, Anal. Chim.

xposed to a phage suspension containing 3.2× 1011 vir mL−1

or 1 h. After incubation with the phage solution, the sensor sur-ace was treated with DPBS and then was placed in a humidifiedhamber at 4 ◦C for 24 h; the sensor surface was then blocked

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Fig. 3. A typical representative dose responses obtained from a phage immobi-lized SPR sensor in a flow through mode. Curve A represents responses obtainedfrom the working phage, 1G40 immobilized in one of the two channels of thesensor against graded concentrations of �-gal (0.0032–210 nM). B representsthe responses obtained from the wild-type phage, F8-5 immobilized on the sec-ond channel. Even at the highest concentration of �-gal tested (210 nM), thertw

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ith BSA as described in [1] before tests with sequential con-entrations of �-gal (0.0032–210 nM) began.

.2. Specificity of binding

Specificity of phage binding to �-gal was examined throughwo ways. In the first method, both �-gal specific phage 1G40nd wild-type F8-5 reference phage were separately immo-ilized on the sensor’s two channels, forming working andeference channels. After phage immobilization, both channelsere exposed with graded concentrations of �-gal.Specificity was also re-affirmed via another method, where

ifferent concentrations of phage suspensions (2.2× 1012–.36× 107 vir mL−1) were at first pre-incubated with 22 nM of-gal for 1 h before being flown over the phage immobilized sen-or surface. This restricted the amount of free �-gal available forinding to the immobilized phage on the sensor chip. As the freehage concentration decreased, more �-gal is available to bindo the sensor immobilized phage.

. Results and discussion

.1. Real time responses from the SPR sensor

Fig. 2a shows the real time responses obtained from the SPRensor from the immobilization of both the working phage andild-type phage, followed by a buffer wash and subsequentlocking of the unoccupied gold surface using a 0.005% Tweennd BSA (1 mg mL−1) as the blocking solution and finally run-ing a buffer wash. Fig. 2b represents a real-time detection of-galactosidase by the selected phage immobilized on the sensor

Please cite this article in press as: V. Nanduri et al., Highly sensitive phagActa (2007), doi:10.1016/j.aca.2007.02.071

urface. Wild type F8-5 was used as a control phage to show theesponses due to non-specific binding of enzyme to the sensorurface. The signal on Fig. 2b was normalized to the baselinendicated by arrows in Fig. 2a.

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ig. 2. (a) Real time responses obtained from an SPR sensor when exposed to the dhe time when various solutions were introduced, A: DPBS; B: working (1G40) andepresent a real-time detection of �-galactosidase by the selected phage immobilizedesponses due to non-specific binding of enzyme to the sensor surface. The signal on

esponse obtained from the working phage is approximately ten times morehan that obtained from the wild-type phage indicating the strong affinity of theorking phage to the target antigen, �-gal.

.2. Specificity of binding

Specificity studies showed that the phage 1G40 was spe-ific to the �-gal. Fig. 3 shows the typical representative doseesponse obtained from the working phage and wild-type phage.rom the responses obtained, it can be seen that the workinghage is specific to �-gal and at even the highest concentration of

e-based biosensor for the detection of �-galactosidase, Anal. Chim.

10 nM of �-gal, the responses obtained from the working phageas approximately 10 times greater than that obtained from theild-type phage and thus clearly indicating the specificity of theG40 phage to �-gal.

ifferent solutions used in the phage immobilization protocol. Letters indicateControl phage (F8-5); C: BSA and D: TBS-Tween 20 (0.5%, v/v). (b) Figureon the sensor surface. Wild type F8-5 was used as a control phage to show the(b) was normalized to the baseline indicated by arrows in (a).

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Fig. 4. Specificity of phage. Dose responses of the phage immobilized sensorto �-gal (22 nM) incubated free phage (8.4× 105–2.2× 1011 vir mL−1) priort(r

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Fig. 5. A full range dose response curve. The smooth curve A representsresponses obtained from the working phage, 1G40 immobilized in one of the twochannels of the sensor against graded concentrations of �-gal (0.0032–210 nM)and is the sigmoid fit to the experimental data (� – χ2 = 6× 10−10, R2 = 0.99).Each experiment was replicated twenty times. Experimental values wereobtained by averaging of about 120 data points of each steady-state level ofresponse-time curves. Insert B: Hill plots of binding isotherms showing the ratiooTR

sepossible explanation could be a different packaging and ori-entation of the phage on the surface in course of flow and batchdeposition.

Fig. 6. A dose response curve obtained when using the flow through modeof delivery conducted in a parallel, comparative study with the batch modestudies. The smooth curve A representing phage binding is the sigmoid fit tothe experimental data ( -χ2 = 5.8× 10−10, R2 = 0.99). Data points plotted arethe mean of six experiments, each of which represents a steady state level of

o exposure. The smooth curve is the sigmoid fit to the experimental data– χ2 = 3.5× 10−9, R2 = 0.97). Data points represent steady state level of

esponse–time curves.

Responses obtained from the second method for reaffirm-ng the specificity of the 1G40 phage, showed that at higheroncentrations of free phage, (Fig. 4) pre-incubated with �-al, low binding is observed as a result of low availability of-gal (due to cross interaction with free phage in solution)

o interact with the phage immobilized on the sensor surface.owever, as opposed to that, a higher signal is observed at

ower concentrations of free phage, where more �-gal is avail-ble to interact with phage immobilized on the sensor surface.his availability or non-availability of �-gal in free solution

mplies that the selected phage 1G40 shows specific bindingo �-gal. For these specificity experiments, the lower end ofetection of �-gal binding was chosen, so as to avoid anyossibility of external noise signal that may occur at higheroncentrations.

.3. Binding studies

Binding of �-gal to immobilized phage showed variationsepending upon the condition of the surface of the sensors andhe different experimental conditions under which binding stud-es were conducted. The two major experimental setups, flow

ode and batch mode that were used to study binding pro-uced different results. In the flow through mode, two sets ofxperiments were conducted. To analyze the characteristics ofow through mode alone, 20 experiments were conducted under

dentical conditions so as to ensure consistency and repeatabil-ty of results obtained (Fig. 5) In the second set, (Fig. 6) sixxperiments were conducted in parallel to batch mode methodFig. 7) so as to enable comparative studies with batch mode.omparative binding studies conducted using both the flow

Please cite this article in press as: V. Nanduri et al., Highly sensitive phagActa (2007), doi:10.1016/j.aca.2007.02.071

hrough and batch mode system was repeated six times underdentical conditions. Table 1 summarizes the mean Kd, bind-ng valences and Hill coefficients obtained for flow throughnd batch mode of delivery of solutions to the gold SPR

roTR

f occupied and free phages as a function of �-galactosidase concentrations.he straight line is the linear least squares fit to the data (slope = 0.61± 0.04;= 0.98).

ensor surface. This shows that flow through mode is moreffective in alleviating the sensitivity of the SPR sensors. A

e-based biosensor for the detection of β-galactosidase, Anal. Chim.

esponse-time curves. Insert B: Hill plots of binding isotherm showing the ratiof occupied and free phages as a function of �-galactosidase concentrations.he Straight line is the linear least squares fit to the data (slope = 0.59± 0.04;= 0.99).

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Fig. 7. Typical binding mean responses of �-galactosidase to phage immobilizedolR

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n the gold surface using batch mode of delivery, repeated six times. The straightine is the linear least squares fit to the data (slope = 2.6× 10−4± 1.5× 10−5;= 0.99).

.4. Phage deposition and surface coverage

The thickness, the orientation, and the surface coverage ofhe phage layer through physical adsorption play a vital rolen increasing the efficiency of the sensitivity of the sensor andlso on the manner of interaction with the target analyte (�-gal).ung et al. provided a mathematical formalism for understandinghe SPR signals from adsorbed layers using a variety of struc-ures [28]. Liedberg et al. proved the exponential decay of SPResponse with distance from the surface [29]. Lukosz proved lin-ar and nonlinear relationships between variations in effectiveefractive indices and thin and thick adlayer thickness, respec-ively [30,31]. Specifically, the thickness of the phage adlayern the sensor surface was calculated using equations describedy Naimushin et al. [26] and Jung et al. [28],

a =(

ld

2

)(neff − nb)

(na − nb)(1)

here da is the thickness of the adlayer, ld is the characteris-ic decay length (307 nm), neff is the effective refractive of thedlayer from SPR signal, nb is the refractive index of buffer1.333), and na is the refractive index of protein adlayer (1.57).ig. 8 shows a typical increase in the adlayer thickness for the

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f the SPR sensor in flow mode and batch mode. The differentverage thickness of phage 1G40 adlayer deposited through flowhrough and batch mode (Table 1) could play a significant role in

able 1omparative responses from flow through and batch modes of immobilization

arameters Flow through modeimmobilization

Batch modeimmobilization

issociation constant Kd (nM) 1.3 ± 0.001 26 ± 0.003inding valency 1.5 ± 0.03 2.4 ± 0.01ill coefficient 0.66 ± 0.002 1 ± 0.002hage adlayer thickness (nm) 3 ± 0.002 0.66 ± 0.001

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hrough physical adsorption using the flow through and batch mode methodss described. The adlayer thicknesses for the two modes are summarized inable 1.

he orientation and conformation of binding of the phage-� galomplex. The higher binding valency in the batch mode studiesould be attributed to a higher availability of binding sites on thehage layer, as a result of the comparatively thinner depositionf the phage adlayer and thus resulting in a comparatively moreexible conformation for multi-valent binding as opposed to aore rigid, thicker and tighter packing during the flow throughode. The corresponding mean responses of �-gal binding to the

hage show a sigmoidal dose response for flow through mode,s seen in Fig. 6(A) as opposed to a linear response (Fig. 7)btained from batch mode. The responses shown for both theow through and batch mode experiments were the mean of sixxperimental data repeated under identical conditions. The dif-erences between adlayer thicknesses for two modes may be dueo very different manner of phage supply to the surface of theensor, and thus, very different pattern of the phage layer. Theontinuous flow mode with flow turbulences allows an “active”istribution of the phage rod-shaped molecules along with flowirection. Meanwhile, in batch mode distribution the phage bind-ng occurs in “static” conditions, when rod-shape phage is lefto its own and binding takes place chaotically, with more dif-usional problems and in longer period of time. This leads toifferent shaping/orientation of the phage on the surface, andherefore, to different kinetic characteristics of the binding. Theower value of the adlayer in the batch mode experiments wherehe responses show a linear relationship in comparison to thosehere flow through mode was employed agrees well with earlier

tudies [30,31].

. Conclusions

The studies show that phage can be used as the sensing probe

e-based biosensor for the detection of �-galactosidase, Anal. Chim.

or the specific and sensitive detection of �-gal. Employing theow through mode gives us a sensor of higher sensitivity inomparison to that obtained through batch mode studies. On thether hand, the binding valences were higher in the batch mode

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tudies as opposed to the flow through mode. This was possiblyue to divalent interaction of phage-�-gal which could be as aesult of:

a) The thin nature of the phage adlayer which could in turn,increase the flexibility of the phage layer making more bind-ing sites available to �-gal.

b) Due to the stabilization of the phage layer prior to beinginteracted with �-gal.

c) Or the combination of both the above factors.

cknowledgments

Support for this work comes from NSF Grant CTS-330189, DAADOJ-02-C-0016, Aetos Technologies Inc., androm Auburn University Detection and Food Safety CenterUSDA Grant). Help from Dr. Jerry Elkind and Dr. Dwightartholomew are greatly appreciated. Acknowledgments arelso due to Dr. Valery A. Petrenko, for graciously providinghage for this study.

eferences

[1] V. Nanduri, A.M. Samoylova, V.Petrenko, V. Vodyanoy and A.L.Simonian,Comparison of optical and acoustic wave phage biosensors, 206th Meetingof The Electrochemical Society, Honolulu, Hawaii, October 3–8, (2004).

[2] V.A. Petrenko, V.J. Vodyanoy, J. Microbiol. Methods 53 (2003) 253.[3] J. Abad, M. Pariente, F. Hernandez, L. Lorenzo, E. Anal. Chim. Acta. 368

(1998) 183.[4] V.A. Petrenko, G.P. Smith, X. Gong, T. Quinn, Protein Eng. 9 (1996)

797.[5] B. Serra, M.D. Morales, J. Zhang, A.J. Reviejo, E.H. Hall, J.M. Pingarron,

Anal. Chem. 77 (2005) 8115.

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