A novel low-cost and easy to develop functionalization platform. Case study: Aptamer-based detection...

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Talanta 80 (2010) 2157–2164

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A novel low-cost and easy to develop functionalization platform. Case study:Aptamer-based detection of thrombin by surface plasmon resonance

Cristina Polonschii a, Sorin Davida, Sara Tombelli b,Marco Mascinib, Mihaela Gheorghiua,∗

a International Centre of Biodynamics, Intrarea Portocalelor 1B, 060101 Bucharest, Romaniab Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy

a r t i c l e i n f o

Article history:Received 4 August 2009Received in revised form 27 October 2009Accepted 5 November 2009Available online 12 November 2009

Keywords:Carboxylated cross-linked BSA filmThrombinAptamerSurface plasmon resonanceSpreeta

a b s t r a c t

A novel low-cost platform to assess biomolecular interactions was investigated using surface plas-mon resonance and an aptamer-based assay for thrombin detection. Gold SPR surface functionalizedwith a carboxylated cross-linked BSA film (cBSA) and commercially available carboxymethylated dex-tran chip (CM5) were used as immobilization platforms for the thrombin binding aptamer. The highend commercial instrument Biacore 3000 and a custom made FIA set-up involving TI Spreeta sensor(TSPR2K23) were used to assess different concentrations of thrombin within the range 0.1–150 nMboth in buffer and in a complex matrix (plasma) using the obtained aptasensors. Based on dataderived from both CM5 and cBSA platforms, the cBSA aptasensor exhibited good selectivity, stabil-ity and regeneration ability, both in buffer and in complex matrices (plasma), comparable with CM5.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Bioaffinity-based methods of detection usually require immo-bilization of one interaction partner (ligand) to the biosensorsurface. Typically the ligand can be a protein or a nucleic acid.As direct physical adsorption to surfaces induces denaturationof the proteins [1], an intermediate functionalization layer forprotein immobilization is needed. Thus the protein ligand willnot be in direct contact with the surface hence preserving itsbinding activity. Currently there are several functionalization pro-tocols and commercially available platforms for biomoleculesimmobilization such as carboxymethylated dextran with vari-ous carboxymethylation levels, or self-assembled monolayer ofthiols, proteins, etc. [2]. The commercial platforms are usuallyexpensive, while less expensive custom preparation protocolsmay require several days. Moreover, the preparation requiresthorough cleaning of the surface with strong oxidizing solutionsthat can damage the surface or, if the case, the plastic hous-ing.

The aim of this study was to investigate the use of the car-boxylated cross-linked bovine serum albumin (cBSA) film as afunctionalization platform for immobilization of biomolecules andits feasibility for surface plasmon resonance assays for throm-

∗ Corresponding author. Tel.: +40 21 3104354; fax: +40 21 3104361.E-mail address: [email protected] (M. Gheorghiu).

bin quantification in complex media. The cBSA film was selecteddue to its carboxyl groups which allow further immobilizationof biomolecules via amino-coupling chemistry and its BSA back-ground providing good blocking ability of the gold surface. As areference we used the CM5 sensor (Biacore AB, now GE Health-care), a carboxymethylated 3D dextran matrix, which is a generalpurpose chip for immobilization of a wide range of ligands, fromsmall organic molecules to proteins, nucleic acids and carbohy-drates. The cBSA and CM5 functionalization platforms immobilizedwith the thrombin binding aptamer were investigated compar-atively in terms of thrombin detection using the Biacore 3000.Furthermore, the cBSA platform was also implemented on a low-cost, versatile Spreeta (Texas Instruments, USA) based SPR systemfor aptamer immobilization and thrombin detection in buffer and inplasma.

Thrombin and its specific aptamer were selected for this studynot only because this bioaffinity pair is well studied, but alsobecause thrombin detection and quantification in complex biolog-ical samples such as serum or plasma is clinically relevant anddevelopment of an easy to use and cost-effective measurementsystem is of great interest. Blood coagulation conventional assaysare usually indirect because they measure the clotting fibrinogen,which also depends on other factors [3].

Thrombin (coagulation factor II) is a highly specific serine pro-tease of trypsin-like specificity that converts soluble fibrinogen intoinsoluble strands of fibrin. This protein which plays an importantrole in thrombosis and hemostasis exhibits both enzymatic and

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2158 C. Polonschii et al. / Talanta 80 (2010) 2157–2164

hormone-like properties that can be both pro- and anti-coagulant.Therefore, thrombin is a key element in various pathogenesis suchas leukemia, arterial thrombosis, liver disease, etc. [4] and regulatesnumerous processes in inflammation and tissue repair at the vesselwall [5].

Thrombin also has hormone-like properties—it can regulate cellsignaling by cleaving and activating receptors [6] and it binds withand activates blood cells (except erythrocytes), endothelial cells,fibroblasts, etc. [7].

Thrombin concentration in blood can vary significantly: undernormal conditions thrombin is present in blood in its inactiveform (prothrombin) and during the coagulation process can reachlow micromolar concentrations. Low nanomolar levels of throm-bin generated when hemostasis is initiated are also important tothe entire process. Thrombin has also been detected at high pico-molar range in blood of patients with coagulation abnormalities[8].

Aptamers are oligonucleic acid molecules that bind to a specifictarget molecule and are created by in vitro selection from a largerandom sequence pool by the SELEX (Systematic Evolution of Lig-ands by Exponential Enrichment) process. They are highly specifictowards their molecular targets, which range from small inorganicand organic substances to proteins and even cells [4]. Althoughaptamers are equivalent to antibodies, they possess numerousadvantages such as ease of synthesis, thermal stability and lackof immunogenicity. Also, they are designed in vitro, without theneed for an animal host. Once selected, aptamers are readily pro-duced with high reproducibility and purity by chemical synthesis.Moreover, an aptasensor can be used in a wide variety of sam-ple matrixes, including non-physiological buffers and temperatureconditions that would cause denaturation of typical antibodies[9].

For this study the DNA thrombin binding aptamer 15-mer 5′-GGTTGGTGTGGTTGG-3′ carrying a 20-mer poly thymine spacerwas chosen because reported results indicated best performancesin comparison with other available sequences. The spacer keepsthe aptamer distant from the sensor surface, thus avoiding sterichindrance and allowing the proper conformation for molecularrecognition [8].

The 15-mer thrombin binding aptamer was the first one selectedin vitro, specific for a protein without nucleic acids-binding prop-erties [10] and has a high specificity for the fibrinogen bindingexosite of thrombin. This oligonucleotide is in equilibrium betweena random and a folded state, equilibrium which is shifted towardsthe four-stranded structure by binding to thrombin. The throm-bin aptamer undergoes a transition into a G-quadruplex structureupon binding thrombin [11] and restricts its activity [12]. The 15-mer thrombin binding aptamer is the most extensively investigatedone although several other aptamers were also selected, includinga 29-mer one specific to the heparin binding exosite of thrombin[13].

In bioaffinity assays, the aptamer immobilization to the surfaceis crucial. Thus, an ordered and oriented layer must be obtained toensure the flexibility of the bioreceptor without altering the affinityfor the target molecule.

Modifications induced at the biofunctionalized surface of trans-ducers as result of specific interactions with target analytes aredetected by various biosensing methods. In the present studywe use surface plasmon resonance (SPR), an optical techniquethat enables real-time monitoring of changes in the refractiveindex of a thin film close to a surface (up to 150 nm distancefrom the surface) as a result of biomolecular interactions [14].In the last years, SPR has matured into a powerful, well estab-lished, biosensing technique, providing real-time kinetic infor-mation on molecular binding and continuous process monitoringcapabilities.

2. Experimental

2.1. Chemicals and reagents

Human �-thrombin, bovine serum albumin (BSA), streptavidin,avidin, human IgG, biotin, N-hydroxysuccinimide (NHS), 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and ethanolaminewere purchased from Sigma–Aldrich (Germany).

Human plasma (without fibrinogen) was provided by theNational Institute of Transfusional Hematology “Prof. Dr. C.T. Nico-lau” (Bucharest, Romania).

All the other reagents for buffers were purchased from Sigma,except for the Surfactant P20 which was provided by Biacore AB(Sweden).

The buffers used for the experiments are: phosphate buffersaline solution (PBS), pH 7.4 containing 137 mM NaCl, 10 mMphosphate, 2.7 mM KCl; streptavidin immobilization buffer, 10 mMacetate buffer pH 5; avidin immobilization buffer, 10 mM acetatebuffer pH 4.5 containing 0.125 M NaCl; aptamer binding buffer,300 mM NaCl, 20 mM Na2HPO4, 0.1 mM EDTA, pH 7.4 [8]; thrombinrunning buffer: 50 mM Tris–HCl, pH 7.4, 140 mM NaCl, 1 mM MgCl2[8]; streptavidin/avidin running buffer: HBS-EP buffer (10 mMHEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20).

Succinic anhydride and sodium hypochlorite (NaOCl) were sup-plied by Acros Organics (Belgium).

The biotinylated 15-mer thrombin binding aptamer (TBA) withpolyT(20) tail used in this study having the following sequence: 5′-biotin-TT TTT TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3′ waspurchased from MWG Biotech.

Deionized water (Millipore) was used throughout the prepara-tions. All chemical reagents were of analytical grade and were usedwithout further purification.

2.2. Sensor chips

CM5 (Biacore AB, now GE Healthcare).Plain gold unmounted chips (Ssense, The Netherlands).

2.3. Equipments

2.3.1. Biacore 3000Comparative CM5 and cBSA measurements were performed

using the Biacore 3000 instrument (Biacore AB, now GE Health-care). The SPR signal provided by the instrument is a direct measureof the angle of minimum reflected intensity. The unit of SPRresponse (resonance unit, RU) is an arbitrary unit chosen so that1 RU corresponds to a shift in angle of 10−4 degrees, which in turncorrelates with a change in refractive index of about 10−6 [15].

2.3.2. Custom made cost-effective SPR systemA dual channel SPR system based on the Spreeta TSPR2K23 SPR

sensor (Texas Instruments, TX, USA) has been developed withinInternational Centre of Biodynamics, Bucharest, Romania.

A two channels polydimethylsiloxane (PDMS) flow cell ismounted on the Spreeta sensor and the liquid sample is deliveredover the sensor surface by the automated fluidic system comprisedof syringe pumps and valves actuated by computer. The flow chan-nels are 8 mm long by 0.8 mm wide with a height of 150 �m. Eachflow channel is connected to an independent flow circuit controlledby a syringe pump, allowing simultaneous, as well as independentanalyses. The flow rate range is 40–1000 �l/min with incrementsof 1 �l/min. Sample injection is achieved using electrically actu-ated injection valves, one for each flow circuit. The sample loopsof the injection valves are loaded with the desired sample througha selection valve. Sample loading, injection and washing protocolsare preprogrammed and the user only selects the measurement


C. Polonschii et al. / Talanta 80 (2010) 2157–2164 2159

Scheme 1. Preparation of cross-linked protein film.

flow channel, the sample volume and flow rate for both buffer andsample. Data acquisition and processing is realized with a LabViewcontrolled interface. The signal received from the SPR sensor is pro-cessed through various software routines to subtract backgroundand reference data in order to obtain the SPR curve. The SPR min-imum is calculated online by analysis of the SPR curve. This datais converted then to refractive index units, or response units usinga calibration protocol. Both SPR data (as refractive index units orresponse units) as well as the SPR curve for each point can be savedfor further analysis.

2.4. cBSA functionalization procedure

Before functionalization, the gold sensor surface was firstcleaned with 5% NaOCl, thoroughly rinsed with deionized water,ethanol and another deionized water washing steps and finallydried under nitrogen stream.

As presented in Scheme 1, sensor functionalization involves acoupled set of processes:

(a) Formation of cross-linked BSA film: 20 �l of 0.4 M EDC weremixed with 20 �l of 0.1 M NHS and 50 �l of a freshly preparedsolution of 5 mg/ml BSA in PBS pH 7.4 and allowed to incubateat room temperature for 5 min. On a horizontally aligned sur-face, 10 �l of the mixture were spread evenly to cover the wholesensor surface. Following a 15 min reaction time at room tem-perature, the sensor was rinsed with 20 mM HCl and deionizedwater and dried under nitrogen stream. The integrity of the filmwas checked using atomic force microscopy (data not shown)revealing a good sensor coverage.

(b) Adding carboxylic groups to the film. Freshly prepared suc-cinic anhydride solution: 0.2 g succinic anhydride in 500 �lDMSO were thoroughly vortexed/sonicated until fully dis-solved. 50 mM Na2HPO4 was added to make up to a final volumeof 10 ml. The pH was adjusted to 6.5 using 5 M NaOH. The BSAcoated plain gold sensors were incubated overnight at roomtemperature in the succinic anhydride solution [16].

2.5. Aptamer immobilization

The same protocols were used for the immobilization/bindingof the biotinylated aptamer on both CM5 and cBSA chips.

Either streptavidin or avidin was first immobilized on the sur-face. The immobilization procedure was performed at a constantflow rate of 5 �l/min and at 25 ◦C. The surface was activated 7 minfor streptavidin and 2 min for avidin using 1:1 volumetric mixturesof 50 mM NHS and 200 mM EDC, followed by a 20 min injectionof 200 �g/ml streptavidin and 50 �g/ml avidin, respectively, and7 min blocking with 1 M ethanolamine hydrochloride pH 8.5.

Before the immobilization, the biotinylated aptamer was heatedat 90 ◦C for 1 min to unfold the DNA strand and then cooled in ice for10 min in order to block the DNA in its unfolded structure [8]. The

thermal treatment unfolds the DNA strand, making the biotin labelat the 5′-end available for the interaction with streptavidin/avidinon the surface.

The thermally treated biotinylated aptamer (1 �M) was injectedfor 7 min, 5 �l/min over the chip surface.

A reference channel immobilized with streptavidin/avidin andblocked with biotin and BSA was used throughout the experiments.

3. Results and discussion

3.1. Biacore 3000 measurements

Several concentrations of thrombin in the physiologically rele-vant range (0.1–150 nM) in both buffer and in plasma were injectedover the sensors surface. All injections were performed at a constantflow rate of 5 �l/min and at constant temperature, 25 ◦C, with aninjection time of 20 min.

The two platforms, CM5 and cBSA, were analyzed using theBiacore 3000 considering: the immobilized TBA surface coverage,the response measured for the same concentration of thrombin,the non-specific response when injecting thrombin in plasma, thebaseline evolution after injection–regeneration cycles, the derivedaverage coefficient of variation (CV) and limit of detection (LOD).

3.1.1. Quality of the cBSA filmBefore avidin/streptavidin immobilization and aptamer bind-

ing, the degree of carboxylation of the cBSA film was evaluated byassessing the charge pre-concentration effect [16]. At pH valuesabove 3.5 the carboxylic groups of the functionalization layer arenegatively charged and due to electrostatic attraction the positivelycharged ligands (in diluted solutions 10–50 �g/ml or less) will beconcentrated on the surface. As this mechanism is suited for mostproteins and several other biomolecules [17] it provides, with lim-ited reagent consumption, a direct evaluation of the reactive siteson the surface.

The pre-concentration test was performed by injecting 50 �g/mlhuman IgG in 10 mM acetate buffer pH 5.5. The response was high(∼5700 RU) indicating that the carboxylated surface was properlyformed. A higher ionic strength buffer at neutral pH (HBS) wasused as running buffer so that the non-covalently bound ligand waswashed off the surface after injection. A short pulse (1 min) of 1 Methanolamine hydrochloride pH 8.5 was used leading to removalof all the remaining traces of electrostatically bound ligand.

The same test was performed for a cross-linked BSA film whichwas not previously treated with succinic anhydride (ncBSA). Theresponse was significantly lower (∼2000 RU).

The non-specific binding of cBSA and ncBSA was compared witha bare gold surface. The non-specific binding of human IgG directlyto the gold surface is not negligible: when 50 �g/ml antibody solu-tion was injected over the bare gold sensor a response of ∼3000 RUwas recorded. Yet, for the bare gold sensor surface, in contrast withthe cBSA and ncBSA surfaces, regeneration with a short pulse of



Table 1Immobilization results for the CM5 and cBSA platforms.

Platform Protein Proteinbound (RU)

TBA bound(RU)

Protein bindingcapacity (RU)

TBA bindingstoichiometry

TBA surface coverage(ng/mm2)

TBA surface coverage(molecules/cm2)

CM5 Streptavidin 3850 1400 817.7 1.71 1.12 6.01E+12Avidin 5060 1370 846.9 1.62 1.09 5.89E+12

cBSA Streptavidin 2760 800 586.2 1.36 0.64 3.44E+12Avidin 1600 415 267.8 1.55 0.33 1.78E+12

1 M ethanolamine hydrochloride pH 8.5 had little effect on thesignal, suggesting that most of the protein remained bound non-specifically.

3.1.2. Immobilized TBA surface coverageSince the SPR response is directly proportional to the mass

concentration of material at the surface, assuming a 1:1 bindingstoichiometry, the analyte binding capacity of a given surface isrelated to the amount of ligand immobilized as follows:

analyte binding capacity (RU) = analyte MW/ligand MW ×immobilized ligand (RU) [17]. Thus the binding stoichiometry of theaptamer to the surface for CM5 and cBSA was calculated:

binding stoichiometry = analyte bound (RU)/analyte bindingcapacity (RU) for 1:1 stoichiometry.

The surface coverage of immobilized aptamer molecules for Bia-core was estimated considering that 1000 RU = 0.8 ng/mm2 [18].

TBA binding stoichiometry had similar values for CM5 and cBSAfor both streptavidin (1.71 and 1.36, respectively), and avidin (1.62and 1.55, respectively), yet TBA surface coverage for CM5 in com-parison to cBSA, was from double, for streptavidin, to triple, foravidin (Table 1). These results are explained due to higher sur-face coverage of binding sites in the 3D carboxymethylated dextranmatrix in respect to the “two-dimensional” cBSA.

When several cBSA aptasensors were immobilized with strep-tavidin/avidin, TBA binding stoichiometry was similar (1.44 and1.36 for streptavidin, 1.55 and 1.43 for avidin,), showing a goodreproducibility in surface functionalization.

3.1.3. Response measured for the same concentration of thrombinin different matrices

For cBSA immobilized with streptavidin and 0.4 ng/mm2 TBAsurface coverage, the response for 100 nM thrombin injection inbuffer was 790 RU, 60% from the response generated with thesame thrombin concentration on CM5 with streptavidin and almosta three times higher TBA surface coverage 1.12 ng/mm2. Wheninjecting 25 nM thrombin in plasma on the same cBSA-streptavidinplatform (Fig. 1A), the response was 450 RU, 70% from the responsemeasured for the same concentration on the analogous CM5-streptavidin platform.

Again the differences are to be explained due to higher surfacecoverage of binding sites in the carboxymethylated dextran matrixin respect to the cBSA.

While similar to the ones indicated by [19] based on a custommade set-up, the dissociation phase for both CM5 and cBSA plat-forms is faster when compared to the same type of experimentsreported [20] on CM5 using the Biacore X instrument. This couldbe explained by the difference between the flow cell volume ofBiacore X (0.06 �l) and of the Biacore 3000 (0.02 �l) used in ourstudy: although the flow rate was the same (5 �l/min), larger wallvelocities are effective in the Biacore 3000 set-up.

3.1.4. Non-specific responseAs the platform is intended to assess target analytes in com-

plex matrices, thrombin spiked plasma samples 1:400 diluted withrunning buffer have been analyzed to check for the non-specificadsorption of proteins (protein content ∼0.2 mg/ml). Thrombin in

plasma determines on the reference channel a non-specific bindingsignal for both CM5 and cBSA aptasensors. For CM5 the non-specificresponse was negligible for streptavidin and around 60 RU foravidin. For cBSA the non-specific response was around 30 RU forstreptavidin and 100 RU for avidin. As expected, due to the lack ofcarbohydrate modification and near-neutral pI [21], streptavidinplatforms show lower non-specific binding as compared to avidinones, with good correlations between CM5 and cBSA.

While the non-specific signal is not negligible (∼30 RU for cBSAwith streptavidin chip), it is nevertheless one order of magnitudelower than the specific signal corresponding to 25 nM of thrombin(see Fig. 1A) and correlates well with the response of the CM5 chip,considered to posses a low non-specific binding potential.

Fig. 1. (A) Injection of 25 nM thrombin in plasma on CM5 and cBSA immobilizedwith streptavidin. (B) Specific versus non-specific signals for cBSA and CM5 uponinjection of 5 �g/ml human IgG.



Table 2Non-specific response when injecting several proteins for cBSA and CM5 platforms.

Protein Concentration cBSA CM5

Non-specific response (RU)

HIgG 5 �g/ml 6.5 ± 2.5 7.5 ± 2.5BSA 1 mg/ml 20 ± 4 19 ± 4Inactivated

thrombin indiluted plasma

0.2 mg/ml 30 ± 2.5 Not tested

In order to investigate the nature of the higher non-specificbinding response for the cBSA sensor, thrombin in plasma mixedwith 150 nM TBA (called in the following “aptamer inactivatedthrombin”) was injected over the cBSA aptasensor. The specificand non-specific responses due to aptamer inactivated thrombinin plasma were the same and also similar with the non-specificresponse measured during regular thrombin in plasma injections,suggesting a significant effect of matrix components on the non-specific binding response.

Additional tests performed with BSA and antibody proteins(human IgG) confirmed a low non-specific binding for concen-trations consistently higher than those of the target compound(Fig. 1B). A summary of the actual values related to different non-specific proteins in HBS running buffer is presented in Table 2.

3.1.5. Baseline evolutionSurface stability and efficiency of the regeneration protocol was

evaluated by monitoring several injection–regeneration cycles.For all surface types the regeneration was performed using a

1 min pulse of 50 mM NaOH, at a flow rate of 5 �l/min.

For thrombin in plasma injected on the CM5 and cBSA platforms,the baseline level after regeneration varied from one measurementcycle to another with a maximum standard deviation value of 5 RUand respectively 8 RU.

To check for the efficiency of regeneration and long term sta-bility, we performed repeated cycles or injection–regeneration.For the CM5 aptasensor, at the end of 9 injection–regenerationcycles, the baseline decreased with 6 RU for the specific channeland with 1 RU for the reference channel for streptavidin (Fig. 2A),and with 30 RU for the specific and 10 RU for the reference chan-nel for avidin (Fig. 2B). For the cBSA aptasensor, at the end of 9injection–regeneration cycles, the baseline decreased with 10 RUfor the specific channel and with 10 RU for the reference channelfor streptavidin (Fig. 2C), and decreased with 22 RU for the spe-cific and increased with 30 RU for the reference channel for avidin(Fig. 2D).

These results show a good efficiency of the regeneration proto-col on both platforms and also a good stability of both platforms.

3.1.6. Calibration curvesCalibration curves consisting of relative response (recorded

after 20 min of injection) versus concentration (for both cBSA andCM5) were fitted with a 4 parameters logistic equation (using BIAe-val 4.1 software for Biacore measurements): f (c) = Rhi − ((Rhi −Rlo)/1 + (c/A1)A2 ), where Rhi = f(c) when c → ∞, Rlo = f(c) when c = 0,A1 is fitting constant 1 and A2 is fitting constant 2.

In Figs. 3A and B the calibration curves for thrombin in plasmaon CM5 aptasensor with avidin and streptavidin are presented,while Fig. 4A and B shows the cBSA aptasensor (with avidin andstreptavidin) responses in respect to injections of thrombin inplasma.

Fig. 2. Baseline variation for each injection–regeneration cycle against initial baseline at the beginning of the experiment for (A) CM5-streptavidin, (B) CM5-avidin, (C)cBSA-streptavidin, and (D) cBSA-avidin.



Fig. 3. (A) Calibration curve for thrombin in plasma on CM5 with streptavidin. (B)Calibration curve for thrombin in plasma on CM5 with avidin.

3.1.7. Coefficient of variation and limit of detectionThe averaged coefficient of variation (CV) was calculated as the

mean of all the coefficients of variation for all the concentrationsconsidered. For CM5 (Biacore) the CV was 3.6% for streptavidin(data not shown) and 2.8% for avidin. For cBSA (Biacore) the CVwas 5.8% for streptavidin and 5.15% for avidin.

The limit of detection (LOD) expressed as the concentration cL

is derived from the smallest measure, xL, that can be detected withreasonable certainty for a given analytical procedure. The value ofxL is given by the equation xL = xbi + ksbi, where xbi is the mean of theblank measures, sbi is the standard deviation of the blank measures,and k is a numerical factor chosen according to the confidence leveldesired [22].

LOD was calculated by the evaluation of the average response ofthe blank plus three times the standard deviation. For streptavidin,LOD for the CM5 platform (1.12 ng/mm2 TBA surface coverage) wasevaluated at 2.5 and 3 nM for cBSA (0.64 ng/mm2 TBA surface cover-age). In the case of avidin, LOD was 5 nM for CM5 (1.09 ng/mm2 TBAsurface coverage) and 5.5 nM for cBSA (0.33 ng/mm2 TBA surfacecoverage), similar to the one previously reported for an aptamer-based SPR assay [19].

To increase the sensitivity of the platform, instead of binding ofthe aptamer directly on the gold surface of the SPR chip, an addi-tional, intermediate layer has been considered providing: improvedaccessibility of the binding sites with likely better conformation ofthe recognition element, leading to increased binding potential andbetter blocking of the surface against non-specific binding.

Fig. 4. (A) Calibration curve for thrombin in plasma on cBSA with streptavidin; inthe inset, injection curves for different thrombin concentrations are included. (B)Calibration curve for thrombin in plasma on cBSA with avidin; in the inset, injectioncurves for different thrombin concentrations are included.

Previous results [8] proved a 4-fold increase in the specific signalfor the same concentration of target compound and aptamer forCM5 chip in comparison with bare gold. Upon optimal choice ofthe aptamer linker (20T) the sensitivity was further increased dueto an 8-fold augmentation in the specific signal compared to baregold.

Our present experimental data further support this improve-ment revealing an increased sensitivity when using streptavidin incomparison with avidin (due to the lack of carbohydrate modifi-cation and near-neutral pI of streptavidin). The surface coverage,though lower than for the 3D dextran matrix characteristic forCM5, is optimum for detection as revealed by the similar responsesof cBSA-streptavidin and CM5 respectively. Notably, the surfaceblocking against non-specific binding is efficient (in complexmatrix, the non-specific response was 6.5% of the specific responsefor cBSA and 1.5% for CM5).

3.2. Custom made SPR system measurements

Having in mind that an important goal of the study was to imple-ment a cost-effective immobilization platform for the TBA on theSpreeta-based sensing system, avidin was chosen for immobiliza-tion on the cBSA sensor due to a better cost/performance ratioagainst streptavidin.



Fig. 5. 50 nM thrombin in buffer injection on cBSA aptasensor. Spreeta-based sys-tem measurement.

In order to maintain a similar concentration of the analyte overthe surface as in the Biacore flow cell (2.4 mm × 0.5 mm × 0.02 mm),the flow rate was adjusted accordingly to our system’s flow celldimensions (0.8 mm × 8 mm × 0.15 mm).

The assay was performed using 50 �l/min flow rate and 16 mininjection time (the maximum injection time for this flow ratedue to sample loop volume). A typical thrombin in buffer injec-tion on both specific and reference channel is presented inFig. 5.

When injecting thrombin in plasma, the reference channelshowed a small non-specific binding response, nevertheless, thebaseline after regeneration returned to its initial value before injec-tion (Fig. 6).

The calibration curve generated with thrombin in plasma usingthe same 4 parameters logistic equation is shown in Fig. 7: the signalincreased with the increased concentration of the added analyte.The average coefficient of variation was 6% and a limit of detectionof 10 nM was obtained for thrombin in plasma.

3.3. Further improvements

Even though the performances of the platforms are compara-ble to ones reported in literature [19], there are two aspects thatare worth further investigating in order to improve the LOD. Arecent study [23] showed that thrombin binding capacity increases

Fig. 6. 100 nM thrombin in plasma injection on a cBSA aptasensor on Spreeta-basedsystem.

Fig. 7. Calibration curve for cBSA avidin platform measured on Spreeta sensor.

as the linker length increases from 0 to 5 thymidine nucleotides anddecreases as the number of thymidine residues increases to 20 (cor-responding to the spacer used in our study). Furthermore, recentevidence pinpoint on the important role of cations present in solu-tion on the G-quadruplex formation: this stabilizing conformationcorresponding to aptamer–thrombin binding is not easily formedin the presence of Mg2+ and Ca2+ [24].

Thus using the optimum linker size and the optimum thrombinrunning buffer are possible ways to further increase the sensitivityof the assay.

4. Conclusions

In this study a new bio-immobilization platform based on a car-boxylated cross-linked BSA film was investigated and comparedwith the commercial available CM5 reference chip. We demon-strate that the cBSA platform is suitable for immobilization ofbioaffinity compound and we tested it with streptavidin/avidin anda biotinylated aptamer. In comparison with the CM5 platform, cBSAexhibited similar sensitivity, considering the immobilized TBA sur-face coverage (LOD 2.5 nM for CM5-streptavidin with 1.12 ng/mm2

and 3 nM for cBSA-streptavidin with 0.64 ng/mm2), a similar coef-ficient of variation (3.6% for CM5 and 5% for cBSA) in detectingthrombin in the presence of other interfering proteins (plasma)and proved stable during numerous injection–regenerationcycles.

The low-cost cBSA aptasensor with avidin was successfullyimplemented on the cost-effective Spreeta-based SPR system (aLOD of 10 nM was obtained for thrombin in plasma and CV of6%, similar to the one provided by the cBSA aptasensor measuredwith the Biacore) amenable for portable platforms development[25].

The cBSA platform exhibited good performance when used incomplex media (plasma), the non-specific response being similarto the one obtained with the CM5 platform. This shows the potentialof this immobilization platform as a low-cost analytical tool in con-junction with selected bioaffinity pairs for real samples (complexmatrix) assessment.

The BSA functionalization procedure is a suitable alternativeto self-assembled monolayers formation or carboxymethyldex-tran coatings, for most SPR chips, including the Spreeta sensorwhich, due to its plastic housing, can be easily altered by theorganic solvents or strong oxidizing cleaning solutions (e.g. piranhasolution).



Besides being a low-cost alternative to commercially availableplatforms, the cBSA film provides additional advantages: it is rel-atively easy to employ, the sensor surface does not require harshwashing methods and the procedure can be completed overnight,and is fully automatable. Moreover the cBSA chips can be strippedoff and re-functionalized repeatedly without destroying the goldsurface or other sensor components.

While current performances of the platforms are comparableto ones reported in literature, further optimizations are envisagedbased on selection of appropriate linker size and thrombin run-ning buffer as possible avenues to increase the sensitivity of theassay.

Acknowledgements

The study was supported by Romanian Grant 81028 PN-IIBiosADN project and by EU NMP3-SL-2008-214107 Nanomagmaproject.

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A novel low-cost and easy to develop functionalization platform. Case study: Aptamer-based detection...

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