application of biosensor surface immobilization methods for aptamer

CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 39, Issue 3, March 2011 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2011, 39(3), 432–438.

Received 30 September 2010; accepted 17 October 2010 * Corresponding author. Email: jpwang@ zju.edu.cn This work was supported by the National Natural Science Foundation of China (No.20907041). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60429-X

REVIEW

Application of Biosensor Surface Immobilization Methods

for Aptamer ZHOU Ling, WANG Ming-Hua, WANG Jian-Ping*, YE Zhun-ZhongCollege of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310029, China

Abstract: Aptamer with small molecular weight, simple structure, and easy synthesis, which can be used repeatedly and preserved for long time, has important applications in biosensor field. This article describes several commonly used methods for fixing aptamer onto the sensor surfaces, including the gold-sulfur self-assembled monolayer, covalent bond, biotin/avidin affinity, complementary nucleic acid chain connection, as well as current research progress and the characteristics of each method. Development of aptasensors with wide detection range, low detection limit, short detection time, and capability of detection of multitargets will certainly facilitate its application in food quality and safety testing, disease diagnosis, drug analysis, environment monitoring, etc. Key Words: Aptamers; Biosensors; Fixation methods; Review

1 Introduction

Aptamers are single-stranded DNA or RNA molecules 25–60 base long with high specificity to various ligands such as proteins, small molecules, etc. In contrast to antibodies, aptamers are chemically prepared by in vitro selection procedure developed simultaneously in early 1990s by Ellington[1] and Tuerk[2] laboratories based on Systematic Evolution of Ligands by EXponential enrichment (SELEX). Biosensors based on DNA or RNA aptamers (aptasensors) were first reported by Potyrailo[3] in 1998, and they can detect target proteins in nM successfully. Utilization of aptamers for target recognition instead of antibodies is of particular interest in assay systems because the specificity and affinity of aptamers are equal or superior to those of antibodies[4]. In addition, aptamers have a number of advantages compared with antibodies or enzymes, such as ease of methods for synthetic modification or immobilization[5], a wide range of target molecules containing proteins, nucleic acids, and other inorganicand organicmolecules[6–9], increased heat stability, and tolerance to wide ranges of pH and salt concentrations[10]. Furthermore, unlike antibodies or enzymes, aptamers are

capable of being reversibly denatured, which facilitates capture and release of target compounds in reusable applications. As to these merits, aptamers have been recently used in analytical chemistry applications as immobilised ligands in biosensors’ design or in homogeneous assays.

But, as biologically active molecules, the modification and immobilization of aptamers onto biosensor surfaces will have a certain impact on the excellent properties of aptamers. The integrity of biological activity and conformational change of the fixed aptamers can also affect the performance of aptasensors. This article covered various methods for the immobilization of aptamers onto different substrates (e.g., gold, silicates, and carbon nanotubes) that have been utilized in diverse array of biosensors. Meanwhile, the advantages and disadvantages of each method were systematically described, and potential ways to solve these problems were proposed in most cases. The practical applications in food control, disease diagnosis, drug analysis, environment monitoring, etc., were examined as well.

2 Basic principle of aptasensors Aptasensors for target detection are an analysis device that

ZHOU Ling et al. / Chinese Journal of Analytical Chemistry, 2011, 39(3): 432–438

closely links biological recognition elements to signal conversion elements[11]. Figure 1 shows the schematic structure of aptasensors. The recognition elements–aptamers can be screened by a variety of methods, and the characteristics of process are shown in reference[12]. The signal conversion elements have many species, including electrochemical electrodes, thermal components, semiconductors, optical components (such as optical fiber, and surface plasma), piezoelectric devices (such as quartz crystal microbalance, and surface acoustic wave), and so on. The basic principle of detection is that first the sensitive biological materials (aptamers) should be fixed in solid phases (molecular recognition elements). Then, the immobilized aptamers can specifically bind with target molecules. Finally, the identification signals generated by signal converter can be quantitatively processed into electricity, Optical signal, and can be amplified and output by the instruments, while establishing a linear measurement between the signal changes and target concentrations.

3 Various immobilization methods for fixing

aptamers onto biosensor surfaces 3.1 Direct attachment to gold electrodes

Direct attachment to gold electrodes[13] is that the 3' or 5'

thiol-labeled aptamers self-assemble onto the gold through gold-sulfur bond interaction and form ordered single-carrier membrane. This is the most simple and commonly used method. The biosensor surfaces include electrochemical gold electrodes[14–18], surface plasmon resonance (SPR)[19,20], quartz crystal microbalance(QCM)[21], and other gold-coated plane surfaces as well as gold nanoparticles, gold nanorods, and other nonplanar surfaces.

In 1997, Herne and Tarlov[22] reported a two-step method to immobilize DNA molecules onto gold electrodes and form gold-sulfur self-assembled monolayer. The first step would be funtionalizing the gold electrodes by immersing them in mM concentration of terminal thiol-modified DNA (SH-DNA) in

solution; then, the second step was that the SH-DNA electrodes were immersed into mM of coadsorbent solution (such as mercapto-hexanol (MCH)) that can block the electrode surfaces which were not covered by the target DNA molecules to prevent nonspecific adsorption. In 2005, Xiao et al[14] successfully constructed an aptasensor by the two-step method in which thiol-modified aptamer self-assembled through the gold-sulfur bond onto the gold electrode surfaces. After adding target thrombin, the current changes were detected as a result of the conformational change of aptamers upon specifically binding the targets. The sensor can detect 6.4–768 nM thrombin in 50% diluted blood. The entire testing process took only a few minutes, and this could satisfy the demand of disease diagnosis and drug detection. The key feature of this approach for sensor detection was that the singal generation was dependent on the conformational change of aptamers. But it was unavailable for aptamers that not had a significant conformational change when identifying target molecules. Hansen et al[15] further extended the two-step method into a variety of substances in 2006. They fixed two kinds of aptamers (thrombin aptamer and lysozyme aptamer) on gold electrodes by two-step method to detect thrombin and lysozyme simultaneously. And the detectable concentration range was 0.5–12.5 pM. According to the different affinities of aptamers between quantum dots labeled and not labeled molecules, the biosensor could competitively detect both targets. It was a universal approach for a variety of aptamers and target detection. At the same time, the sensor had the advantages of good selectivity, no significant cross-reactivity, and can meet the requirements for detection of two substances simultaneously, greatly improving the speed and accuracy of protein detection and disease diagnosis. In addition, the two- step method can be applied in the SPR and QCM sensors, and the detection limit can be reached nM to pM of magnitude as well. The fixed and testing process can be seen in references[18–20].

Aptamers not only could self-assemble onto plane sensor surfaces through gold-sulfur bond interaction but also had some good modifications on the gold particles[23–26], gold nanorods[27], and other nonflat surfaces. For example, Huang

Fig.1 Structure diagram of aptasensors


et al[23] immobilized the thiol-labeled aptamer and MCH onto the surface of gold nanoparticles in suspension successively to detect platelet-derived growth factors (PDGF) in 2005.The sensor system was based on color change of detection solution after PDGF were added, the minimum detectable concentration amounted to nM. The sensor was relatively simple, and the detection process can be observed by naked eyes and did not require complex and expensive chemical analysis equipment. But its sensitivity was only nM. The detection signal depended on the PDGF double cross-linking binding sites, so it cannot meet the various trace species detection in complex matrix. To improve the sensitivity of the sensor, Bai et al[24] fixed gold nanoparticles in suspension onto electrodes, and put the thiol-modified aptamers and MCH on the gold nanoparticles in turn as followed by the two-step method. As the ECL signal changed sensitively after adding the target molecules (lysozyme) into detection solution, the detection limit of 1.0 × 10–13 M and detection range of 1.0 × 10–13–1.0 × 10–8 M can be achieved. This immobilization of aptamers can increase the biosensor sensitivity by 6-fold than directly fix to the gold electrode surfaces or nanoparticles alone, indicating that the nanoparticles-modified electrode could amplify the detection sensitivity. The sensor can be used for real-time monitoring of trace lysozyme in egg. In 2009, Zhen et al[27] fixed the aptamers onto the end of nanorods to detect thrombin. The basic principle was that due to the closure agent (cetyltrimethyl-ammonium bromide) blocking the vertical surface of gold nanorods, thiol-labeled aptamer can only be fixed to the end. With the various concentrations of thrombin added to the reaction system, changes of the nanorod structure occurred, and this can be used for qualitative detection of thrombin. While taking advantage of the specific interactions of aptamers and thrombin, the method can also be used to build new one-dimensional bionano- materials to meet the new demand of medical and biological areas. In summary, the immobilization method of direct formation of a gold-sulfur bond self-assembled film onto the electrodes was simple, did not require complex chemical surface modification, and the detection limit of biosensors can reach nM, or even lower. But there were also some shortcomings, aptamers on the gold surfaces often occur in a strong nonspecific adsorption fashion, so there must be coabsorbents (thiol-labeled short carbon chain compounds). And this method can only be applicable for aptamers on gold or gold-plated surfaces, but not for silicons, carbon nanotubes and other surfaces. It would greatly limit the application of aptasensors if can only be constructed by gold-sulfur immobilization method.

3.2 Covalent attachment to chemically-modified biosensor surfaces

Covalent attachment to chemically-modified sensor

surfaces is that chemical groups (such as amino) labeled aptamers interact with the corresponding chemical groups (hydroxyl, carboxyl, amino, etc.) under the driving force of covalent binding of the chemical bond. So, there a layer of ordered film of aptamer will be formed on the sensor surfaces.

In 1998, Potyrailo et al[3] reported an aptasensor for protein detection. The basic principle was that the target aptamer labeled with amino and fluorescein isothiocyanate in 3' and 5' end, respectively, was fixed at [(3-methyl ethylene oxide) propyl] trimethoxysilane and 1,1'-imidazole-activated electron microscope slides. And then the slides were immersed into ethanolamine solution to close the unbound sites. In this way, the addition of 1,1'-imidazole reduced the interference of epoxide for the initial background signal, making the lowest detected concentration of thrombin to 5 nM, the standard deviation less than 4%, and the analysis time less than 10 min. Kim et al[28] reported another way for fixing aptamers onto silicon surfaces. The silicon surfaces were modified by 3-aminopropyl triethoxysilane, succinic anhydride, and treated with Na2CO3 successfully, then covered with an ordered thin film of carboxyl groups. The amino-labeled aptamers were immobilized onto the sensor surfaces through interaction with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide/N-sulfo- succinimide (EDC/sulfo-NHS). The current signal changed according to the amount of thrombin added. So, the sensor can detect 330 pM thrombin in blood samples for real time. However, the amount of cross-linking agent EDC/sulfo-NHS should be added under strict control because the excess of EDC/sulfo-NHS would cause free aptamers to precipitate, and a little amount would reduce sensor signals or even undetectable. Aptasensor described above not only had low detection limit and fast speed of sample analysis, etc., but also did not require complex sample preparation and it can detect the standard samples and real samples. The advantages were far higher than conventional protein analysis methods (immunoassay, electrophoresis, etc.). So, it can be used as important protein analysis tool in complex matrices.In 2005, Bang et al[29] fixed amino-labeled aptamers to the gold electrodes by interaction with 11-mercapto-undecane acid self-assembled on the surfaces with the cross-linking of 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide/N- hydroxyl-succinimide(EDC/NHS). The linear detectable range could be up to 0–50.8 nM and detection limit 11 nM. Compared with the fixation approach in the literature [28], the activation approach significantly reduced the complexity and simplified the immobilization process of the sensor. Zhang et al[30], in order to optimize the sensor performance and further reduce the detection limit, amplified detection signal by a new fixation approach in 2009. First, the gold electrodes were immersed in cysteamine hydrochloride solution and activated by amino groups, and then immersed into the Glutaraldehyde and polyamide dendrimer (PAMAM) solution successfully. Finally, the electrode surfaces with a layer of high-density


aldehyde film were used to immobilize the amino-labeled aptamers. The linear detection range of the sensor was 1–50 nM, and detection limit was 0.01 nM. This showed that the introduction of polyamide dendrimers increased aptamer coverage on the sensor surfaces, amplified detection signal, and further optimized the performance of the sensor to make the trace detection to be achieved.

Aptamers could be covalently attached to the other surfaces in addition to silicon, gold etc. For example, So et al[31] fixed aptamers to single-walled carbon nanotubes easily after activated by imidazole-Tween 20. The biosensor based on single-walled carbon nanotubes can detect 1–100 nM, and the detection limit was 10 nM. The performance was far superior to antibodies as biological recognition molecules in carbon nanotubes-based sensors.

The chemically covalent attachment approach increased the specificity of aptasensors and decreased the interference signal of nonspecific adsorption. Aptamers can be fixed onto the sensor surfaces in a variety of ways, enriched the types and applications of sensors. However, this method not only required complex chemical modification, more complicated steps, but also introduced many new interference factors, such as surfactants, cross-linking agents, and other side effects. Therefore, the optimum reaction conditions of various chemical reagents, aptamers, and targets must be considered.

3.3 Biocoatings

Avidin (or streptavidin) is a biotin binding protein that

contains four identical subunits. Each subunit contains one binding site for biotin. The strength of the noncovalent avidin-biotin interaction could be comparable to antigen/ antibody and ligand/receptor[32]. As early as 1986, Ross et al[33] had reported the changes of buffer concentration and pH, denaturants, and extremes of temperature, and so on would not affect the combination of avidin and biotin. Therefore, the high specificity and affinity between avidin (or its derivatives) and biotin are widely used in the immobilization of aptamers onto the sensor surfaces.

Liss et al[34] built an aptasensor based on gold. First, the quartz crystal was immersed in 3,3'-dimercapto dipropionate II (N-succinimidyl ester) (DSP) to activate the surface for directly conjugating avidin. Then, biotin-labeled aptamers were fixed on the crystal by interacting with avidin. The sensor can detect 10 nM IgE. In 2004, Minunni et al[35] activated the gold film-coated quartz chip by 11-Mercapto- undecanol and Carboxylated dextran to interact with avidin using EDC/NHS. And the biotin-labeled aptamers were attached to avidin-modified quartz chip.The sensor can be used to detect HIV-1 transactivator protein: the linear detection range was 0–2.5 mg L–1, and detection limit was 0.65 mg L–1. In 2007, Porfirieva et al[36] reported that

streptavidins were electrostatically adsorbed onto the poly thiophene thiazole-modified QCM sensor surfaces to fix the biotin-labeled aptamers. The linear range for thrombin detection was 10–100 nM. In 2009, Kim et al[37] reported another activation approach, the 3,3'-dimercapto dipropionate- modified gold electrodes were directly coupled with streptavidin using EDC/NHS to interact with biotinylated aptamers. Experimental results showed that the detection limit of tetracycline was 10 nM. Meanwhile, in order to increase the sensor's detection range, lower detection limits and improve the sensitivity of the sensor, Wei et al[38] copolymerized the streptavidin-modified dendritic DNA molecules and polypyrrole on the electrode surfaces simultaneously to form a layer of conducting film. As each dendrimer molecule can be labeled with 2–4 streptavidins, it can greatly increase the amount of biotin-labeled aptamers on the sensor surfaces. And the sensor can detect the A-Botulinum toxin up to 40 mg L–1. These results suggested that the key of aptamers’ fixation by biotin/avidin approach was how to put the avidin on the sensor surfaces. The different modification methods were mainly depended on the activation groups on the sensor surfaces, and they were further decided by the surfactant, which finally determined the streptavidin connection approach. Therefore, the choice of surfactant was the most important.

The immobilization method of biotin/avidin approach on gold electrodes and quartz crystal chip was the most commonly utilized. In addition, Rodriguez et al[39] fixed the aptamers to the indium tin oxide electrode surfaces to detect lysozyme in 2005. They immersed the electrodes in 2-amino benzoic acid solution contained H2SO4 and scanned 8 cycles at 40 mV s–1 to form a layer of polymers. Avidin was attached to electrodes with EDC/NHS to bind biotinylated aptamers. The sensor can detect 0.2 mg L–1 lysozyme. In 2007, Centi etal[40] used the method in [35] to fix the aptamers on Fe4O3 beads that can detect 5 nM thrombin.

In summary, biotin-labeled aptamers interacted with avidin can be fixed on a variety of sensor surfaces, and the aptasensors can be used to detect various targets, such as proteins, antibiotics, biotoxins, and so on. Specifically, compared with other fixation methods, each streptavidin molecule can bind four biotinylated aptamers. This would increase the amount of aptamers on the sensor surfaces, reduce the incidence of nonspecific adsorption, and improve sensor signal-to-noise ratio. Therefore, the fixation approach should be widely used in disease diagnosis, drug analysis, food quality, safety monitoring, and other fields. But the pH values usually had great influence on avidin which was attached to the sensor surfaces. For example, the pH values that were far lower than the isoelectric point of avidin would lead it to take a lot of positive charges. And there would be great electrostatic interactions between aptamers and avidins, making the superiority of this fixed way not fully realized.


3.4 Hybridization onto biosensor surfaces through partially complementary oligonucleotides

Aptamers’ attachment onto sensor surfaces by hybridization

with partially complementary oligonucleotides is another fixation method. The unlabeled aptamers are hybridized to a short chain of nucleotides which are attached to surfaces in advance.

In 2007, Zhao et al[41] designed a new colorimetric aptasensor. The adenosine aptamers were immobilized on the gold nanoparticles by hybridization with partially complementary oligonucleotides. The nanoparticles would change red due to electrostatic repulsion from much more negative charges on partially complementary double-stranded DNA. When the target molecules–adenosine were added in the reaction system, aptamers will bind the adenosine and dissociate from nanoparticles. At this point, the nanoparticles fused together and changed to purple as the surface of gold nanoparticles reduced negative charges significantly. The entire testing process took only 1 min, and the detection limit was 10 M. Compared with the aptasensors constructed by Huang et al[23], the advantage of colorimetric sensors was that the generated optical signal was not dependent on the conformation change or multibinding sites of aptamers. Thus, the sensor can detect proteins, metal ions, DNA molecules, and other targets with the different kinds of aptamers fixed onto the sensor surfaces. In the same year, Wu et al[42] put gold nanoparticles on electrode surfaces and used the same way to connect aptamers on the immobilized nanoparticles to improve the sensitivity of the biosensor. The difference was that it will cause the change of redox current rather than color. The detection range of the sensor was up to 0.1–10 M, and detection limit 20 nM.

Zhang et al[43] extended the fixation method described above to detect a variety of materials simultaneously. Three different fluorescent-labeled aptamers (adenosine, K+, cocaine aptamers) were attached to gold nanoparticles by hybridization with partially complementary oligonucleotides. When added the target molecules, aptamers would bind the target molecules and dissociate from nanoparticles. Meanwhile, fluorescence can be detected. The sensor had high selectivity, low cross-reactivity, and can detect mM targets simultaneously. Although the sensitivity was smaller than the other sensors, it was possible for high-throughput, real-time and multiple target molecules’ detection, rapid analysis of environmental contaminants, and food components in the complex matrices.

In addition to be fixed onto nanoparticles, there were many reports about gold electrodes[44–47]. For example, Peng et al[44]

connected aptamers to electrode surfaces by hybridization with partially complementary oligonucleotides for quantitative detection of lysozyme. The linear detection range was up to 0.2–4.0 nM and detection limit 0.07 nM. In the same year,

Chakraborty et al[45] proposed the fixation direction of aptamers on gold surfaces. The experimental results indicated that the aptamers with thiol modification at 3' end and attachment to electrode surfaces at 5' end were the best. The reason may be that a different direction would affect the secondary structure of aptamers, thus influencing the interaction with target molecules.

Other sensors such as SPR aptasensor through hybridization with partially complementary oligonucleotides on the SPR chip to detect prion protein was shown in the literature[48]. In addition, Liu et al[49] fixed the aptamers on the MnO2- modified glassy carbon electrode in the same way to detect adenosine. The detection range was 1.0–100 nM. In 2010, aptamers were attached to multiwall carbon nanotubes- modified glassy carbon electrode surfaces as described above by Liu et al[50] to detect thrombin. The detection limit was 0.5 pM and detection range 1–500 pM.

Aptasensors constructed through hybridization with partially complementary oligonucleotides were based such that the added targets would cause complementary double-stranded chains dissociate, which lead to the generation of optical or electrochemical signals. The sensors did not depend on aptamer conformation change generated by the binding target. So it is applicable for a variety of sensors constructed by the way discovered above. However, due to the immobilization method involving annealing and hybridization, it is difficult to control the experimental conditions. At the same time, the partially complementary double-stranded chains (aptamers and short oligonucleotides) had the same electrical charges, and the electrostatic repulsion and steric hindrance would be much larger. Therefore, the physical and chemical properties to optimize the immobilization process of aptamers must be changed.

3.5 Others Aptamers directly attached to gold surfaces are

accompanied by the occurrence of nonspecific adsorption due to the interaction between nitrogen and gold surfaces. Therefore, it is necessary to adopt the coadsorbent reagents to reduce the interference of nonspecific adsorption. But there were some people just to take advantage of this nonspecific adsorption for biosensor construction[51–53]. For example, in 2008, Cho et al[51] just utilized the nonspecific adsorption between aptamers and gold nanoparticles (Au-N bond) to detect thrombin. The Raman signal would change with the varied adsorption amount of aptamers caused by different concentration of thrombin added. And the detection limit was 100 pM. In 2009, Li et al[52] also reported that aptamers were adsorbed on to gold nanoparticles nonspecifically by nitrogen-gold bonds to detect heavy metals Hg2+. And the sensor can detect 10–9–10–4 M Hg2+, the detection limit was 0.6 nM with the good selectivity to Cu2+, Pb2+, and other


heavy metal ions. In addition, in 2007, Alvarez et al[54] constructed a

competitive aptasensor for neomycin B detection, where aptamers were modified onto the electrode surfaces by interaction with neomycin B fixed on the electrodes. That can detect 25–2500 M neomycin B in milk. In 2009, they applied the same modification method to the SPR biosensors[55] and significantly improved the detection sensitivity of neomycin B. The detection range of neomycin B could be up to 10–100 M in milk.

4 Summary and prospect Fixing aptamers on the sensor surfaces was the primary and

key step to aptasensor construction. The various methods would not only affect the excellent properties of aptamers but also influence the performance and accuracy of test results. This article reviewed the advantages and disadvantages of the fixation methods as stated above. And it should be chosen based on the actual situation. Meanwhile, because aptamers had many superior properties to antibodies and enzymes, the aptasensors would be of important application value and practical significance in the food quality and safety testing, disease diagnosis, drug analysis, environmental monitoring, and other areas.

Future research will focus on the following aspects: (1) In-depth study of immobilization process of aptamer onto the sensor surface, and the target-induced spatial conformation change of aptamer, as well as the specific binding sites of aptamers to improve and enrich different kinds of surface modification chemistry and immobilization methods; (2) There was little research about how much percentage of aptamers fixed onto the sensor surfaces. The effect of the fixation process can only be indirectly measured by detection range and detection limit. Therefore, it is necessary to work out a direct calculation of the percentage of aptamers on the sensor surfaces; (3) The combination of aptamers and nanomaterials is a key direction for future development, such as nanoparticles not only serve as solid phases but also as a catalyst used to amplify the detection signal of aptasensors. Currently the application of gold particles in optical aptasensors is very successful, and in future, the nanomaterials will be utilized more widely.

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