Thiol-modified Aptamer Research Articles

Aptamer based hybrid monolithic pipette tips supported by melamine sponge for enrichment of proteins

BackgroundThe convenient preparation and application of functionalized organic-inorganic hybrid monolithic materials have obtained substantial interest in the pretreatment of complex samples by solid-phase extraction (SPE). Compared to the in-tube solid-phase microextraction in fused-silica capillaries, micro SPE in plastic pipette tips have fascinating merits for the easily operated enrichment of trace target analytes from biological samples. However, the poor compatibility of organic-inorganic hybrid monoliths with plastics leads to the rare appearance of commercial hybrid monolithic pipette tips (HMPTs). Therefore, how to synthesize the organic-inorganic hybrid monolithic materials with better extraction performance in plastic pipette tips becomes a challenge. ResultsWe develop a facile and cheap strategy to immobilize organic-inorganic hybrid monoliths in pipette tips. Melamine sponge was employed as the supporting skeleton to in situ assemble amine- and thiol-bifunctionalized hybrid monolithic material via “one pot” in a pipette tip, and gold nanoparticles (GNPs) and thiol-modified aptamer against human α-thrombin were sequentially attached to the hybrid monolith within the HMPTs. The average coverage density of the aptamer with GNPs as an intermediary reached as high as 818.5 pmol μL−1. The enriched thrombin concentration was determined by a sensitive enzymatic chromogenic assay with the limit of detection of 2 nM. The extraction recovery of thrombin at 10 nM in human serum was 86.1 % with a relative standard deviation of 6.1 %. This proposed protocol has been applied to the enrichment and determination of thrombin in real serum sample with strong anti-interference ability, low limit of detection and high recovery. SignificanceThe amine- and thiol-bifunctionalized HMPTs prepared with sponge as the skeleton frame provided a novel substrate material to decorate aptamers for efficient enrichment of proteins. This enlightens us that we can take advantage of the tunability of sponge assisted HMPTs to produce and tailor a variety of micro SPE pipette tips for broader applications on the analysis of trace targets in complex biological, clinic and environmental samples.

Codeposition Enhances the Performance of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors, a minimally invasive means of performing high-frequency, real-time measurement of drugs and biomarkers in situ in the body, have traditionally been fabricated by depositing their target-recognizing aptamer onto an interrogating gold electrode using a "sequential" two-step method involving deposition of the thiol-modified oligonucleotide (typically for 1 h) followed by incubation in mercaptohexanol solution (typically overnight) to complete the formation of a stable, self-assembled monolayer. Here we use EAB sensors targeting vancomycin, tryptophan, and phenylalanine to show that "codeposition", a less commonly employed EAB fabrication method in which the thiol-modified aptamer and the mercaptohexanol diluent are deposited on the electrode simultaneously and for as little as 1 h, improves the signal gain (relative change in signal upon the addition of high concentrations of the target) of the vancomycin and tryptophan sensors without significantly reducing their stability. In contrast, the gain of the phenylalanine sensor is effectively identical irrespective of the fabrication approach employed. This sensor, however, appears to employ binding-induced displacement of the redox reporter rather than binding-induced folding as its signal transduction mechanism, suggesting in turn a mechanism for the improvement observed for the other two sensors. Codeposition thus not only provides a more convenient means of fabricating EAB sensors but also can improve their performance.

An electrochemical aptasensor based on silver-thiolated graphene for highly sensitive detection of Pb2.

The presence of lead ions (Pb2+) in the environment not only leads to environmental contamination but also poses a significant risk to public health through their migration into food and drinking water. Therefore, the development of rapid and effective techniques for detection of trace amounts of Pb2+ is crucial for safeguarding both the environment and biosafety. In this study, an aptamer-based electrochemical sensor was developed for specific detection of Pb2+ by modifying a polylysine (PLL) coated silver-thiolated graphene (Ag-SH-G) nanocomposite (PLL/Ag-SH-G) on the surface of a glassy carbon electrode, which was further modified with gold nanoparticles (AuNPs) for attachment of aptamers (Apt) that specifically recognized Pb2+. The Ag-SH-G particles were synthesized using a one-step in situ method, resulting in significantly enhanced electrochemical properties upon incorporating Ag nanoparticles into the PLL/Ag-SH-G composite. Coating of the covalently or no-covalently bonded Ag-SH-G particles with PLL provides an excellent supporting matrix, facilitating the assembly of AuNPs and a thiol-modified aptamer for Pb2+. Under optimized conditions, Apt/AuNPs/PLL/Ag-SH-G/GCE exhibited excellent sensing performance for Pb2+ with a wide linear response range (10-1000 nM), a low detection limit (0.047 nM) and extraordinary selectivity. The sensor was employed and satisfactory results were obtained in river water, soil and vegetable samples for the detection of Pb2+.

A novel dual-mode aptasensor based colorimetry and electrochemical detection of norovirus in fecal sample

Norovirus is a leading cause of acute gastroenteritis in humans. This paper presents the development of a novel dual-mode aptasensor for detecting norovirus using colorimetry and electrochemical methods. The initial colorimetric method utilizes gold nanoparticles (AuNPs) and sodium chloride to establish a positive correlation between the concentration of norovirus in a solution and the absorbance ratio A650/A520. The naked eye can detect concentrations as low as 0.1 μg/mL, corresponding to a Ct value of 33 (2.2 copies/μL, CT = 34.102–3.2185·lgX), allowing for qualitative and semi-quantitative analysis. For more accurate trace analysis, a gold electrode is modified with a thiol-modified aptamer and closed with 6-Mercapto-1-hexanol. After incubation with norovirus, the virus specifically binds to the aptamer, causing changes in its spatial structure and distance from the electrode surface. These changes can then be detected using electrochemical square wave voltammetry (SWV). Under optimal reaction conditions, the peak current from SWV exhibits a strong linear relationship with the logarithm of norovirus concentrations between 10−9 μg/mL and 10−2 μg/mL. The regression equation Y = 14.76789 + 1.03983·lgX, with an R2 value of 0.987, accurately represents this relationship. The limit of detection was determined to be 1.365 × 10−10 μg/mL. Furthermore, the aptasensor demonstrated high specificity for norovirus in fecal samples, making it a promising tool for detecting norovirus in various sample types.

Aptamer-modified magnetic nanoparticles for extraction of atrazine in environmental water samples

In this work, a novel aptamer magnetic sorbent was prepared and used as magnetic solid-phase extraction sorbent for selective isolation of atrazine (ATZ) in water samples. The magnetic oligosorbent was fabricated by immobilizing DNA aptamer onto the surface of vinylized magnetic nanoparticles (VMNPs) via “thiol-ene” click chemistry reaction. After optimization of different aptamer coupling parameters, the following experimental conditions were found to give best results: 100 mg VMNPs incubated with 1 μmol L−1 thiol-modified aptamer for 5 h at 55 °C. The resulting oligosorbent was characterized by different instrumental techniques such as transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and inductively coupled plasma mass spectrometry (ICP-MS). Then, several parameters of the extraction protocol were investigated to achieve the best ATZ extraction performance. The optimized aptamer-based magnetic solid-phase extraction (Apt-MSPE) protocol followed by HPLC with diode-array detection gave detection and quantification limits for ATZ of 0.005 µg L−1 and 0.018 µg L−1, respectively. The applicability of this protocol was demonstrated by analyzing several environmental water samples from of different sources with recovery values from 85.5 to 94% and relative standard deviations less than 2.5%. Moreover, the developed Apt-MNPs sorbent could be re-used at least 12 times, without losses in its extraction capacity.

Aptamer-functionalized stir bar sorptive extraction for selective isolation, identification, and determination of concanavalin A in food by MALDI-TOF-MS

An aptamer-functionalized stir bar sorptive extraction (SBSE) coating is described for the first time devoted to selective isolation and preconcentration of an allergenic food protein, concavanalin A (Con A), followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) determination. For this purpose, the polytetrafluoroethylene surface of commercial magnetic stir bars was properly modified and vinylized to immobilize a thiol-modified aptamer against Con A via straightforward “thiol-ene” click chemistry. The aptamer-functionalized stir bar was employed as SBSE sorbent to isolate Con A, and several parameters that can affect the extraction efficiency were investigated. Under the optimized conditions, Con A was extracted and desorbed during 30 and 45 min, respectively, at 25 °C and 600 rpm. The SBSE MALDI-TOF-MS method provided limits of detection of 0.5 μg mL−1 for Con A. Furthermore, the SBSE coating was highly selective to Con A compared to other lectins. The developed method was successfully applied to the determination of low levels of Con A in several food matrices (i.e., white beans as well as chickpea, lentils, and wheat flours). Recoveries ranged from 81 to 97% with relative standard deviations below 7%. The aptamer-based stir bars presented suitable physical and chemical long-term stability (1 month) and a reusability of 10 and 5 extraction cycles with standards and food extracts, respectively. The developed aptamer-affinity extraction devices open up the possibility of developing novel highly selective SBSE coatings for the extraction of proteins and peptides from complex samples.Graphical abstract

Hemin-catalyzed SI-RAFT polymerization for thrombin detection

Ultrasensitive protein detection has considerable potential for early diagnosis and screening of diseases. Here, based on Hemin-catalyzed surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization, this work developed an assay for ultrasensitive fluorescent biosensing of proteins and established a thrombin fluorescent sensor. First, the amino-modified aptamer 1 was immobilized on Fe3O4 magnetic beads, and then thrombin was added to form the aptamer 1-thrombin bioaffinity complex. Another thiol-modified aptamer 2 binds to bound thrombin to form a sandwich structure. Fluorescein O-methacrylate was used as the monomer, acetylacetone (ACAC) as the initiator, and Hemin as the catalyst. Through Hemin-catalyzed SI-RAFT polymerization, a large number of thrombin molecules are labeled with fluorescein O-methacrylate, resulting in severalfold amplification of the fluorescent signal. By detecting the fluorescent signal in thrombin from 1.0 fM to 0.1 nM, the proposed super sandwich strategy can detect thrombin at 0.98 fM, which is lower than the detection limit of most other methods.

Au Nanoparticle-Based Surface-Enhanced Raman Spectroscopy Aptasensors for Paraquat Herbicide Detection

Herein, we developed a surface-enhanced Raman spectroscopy (SERS)-based aptasensor platform by modifying gold (Au) nanoparticle SERS substrates with a thiol-containing aptamer for sensitive and selective detection of paraquat (PQ), a commonly used herbicide for weed and grass control. The Au nanoparticle SERS substrate used in this study is a commercial SERS substrate (ONSPEC-Lite, https://www.nectec.or.th/en/innovation/product-innovation/onspec-series.html) developed by our group at NECTEC, Thailand. The average size of the nanoparticle is 59 ± 17 nm. A thiol-modified aptamer PQ77-SH was immobilized on the Au nanoparticle surface and used as a bioreceptor for binding the PQ molecules selectively on the SERS substrate. PQ detection in water medium using the SERS-based aptasensor substrates was carried out by observing the Raman peak intensities of PQ at different concentrations and compared with SERS substrates without aptamer modifications. Modification of the SERS substrates with aptamers improves the sensitivity and enables selective detection of PQ with a limit of detection estimated to be 0.10 ± 0.03 μM. The developed SERS-based aptasensor platform was evaluated using natural water samples collected from a nearby pond and demonstrated good stability against interferences from other similar herbicides and insecticides present along with PQ in water. The findings of this study indicate that the SERS-based aptasensor not only increases the sensitivity of SERS substrates but also, and this is crucial, makes them selective toward PQ detection, assisting in resolving the problem of low selectivity from conventional SERS substrates.

Development of aptamer based colorimetric assay for whole blood glycated hemoglobin (HbA1c) estimation using gold nanoparticles

Thiol-modified aptamer oligonucleotides containing gold nanoparticles (G@NPs) bound to glycated hemoglobin (HbA1c) with high affinity in whole blood samples. Aptamer thiol groups enhanced the stability of aptamers adsorbed on the surface of G@NPs. A detection strategy was used to visually confirm the presence of HbA1c and the absorbance of the colored compounds at selected wavelengths. Modified G@NPs are used in many colorimetric detection strategies due to their high extinction coefficient and strongly distance-dependent optical properties. In the present study, as the target analyte reports a method to prepare gold nanoparticle-bound purple aptamer aggregates that rapidly collapse into bluish red dispersed nanoparticles upon binding of HbA1c. HbA1c was detected in unpretreated whole blood after dilution at pH 7.4 and with UV-VIS double beam spectrophotometer with a lower limit of detection (LOD) (0.1 μM) and a much broader linear detection range (0.1 μM–100 μM). This current colorimetric method provides a rapid (7 min), accurate and high determination of whole blood HbA1c with minimal interference from uric acid, bovine serum albumin, hemoglobin, and glucose. The lower detection limit and broader linearity of aptamer-bound G@NPs hybrids improve the accuracy and precision of HbA1c measurements.

Preparation of a Highly Sensitive Electrochemical Aptasensor for Measuring Epirubicin Based on a Gold Electrode Boosted with Carbon Nano-Onions and MB.

Epirubicin is prescribed as an essential drug for treating breast, prostate, uterine, and gastrointestinal cancers. It has many side effects, such as heart failure, mouth inflammation, abdominal pain, fever, and shortness of breath. Its measurement is necessary by straightforward and cheap methods. The application of aptamer-based electrochemical sensors is accounted as a selective option for measuring different compounds. In this work, a thiol-modified aptamer was self-assembled on the surface of the gold electrode (AuE) boosted with carbon nano-onions (CNOs), and coupled with methylene blue (MB) as an electroactive tracker to achieve a sensitive and selective aptasensor. In the absence of the epirubicin, CNOs binds to the aptamer through a π-π interaction enhancing the MB electrochemical signal. When epirubicin binds to the aptamer, the adsorption of CNOs and MB to the aptamer is not well established, so the electrochemical signal is reduced, consequently, the epirubicin value can be measured. The prepared aptasensor demonstrated an excellent sensitivity with a curve slope of 0.36 μI/nM, and 3 nM limit of detection in the linear concentration range of 1-75 nM. The prepared aptasensor was accurately capable of measuring epirubicin in blood serum samples.

Highly sensitive detection of aflatoxin B1 by in situ growth of electroactive polymers via ring-opening metathesis polymerization

In this study, an electrochemical sensor was fabricated for detecting aflatoxin B1 (AFB1) based on in situ-initiated ring-opening metathesis polymerization. The thiol-modified aptamer was first immobilized on the Au electrode through Au-S bonding to capture AFB1. Then, the acrylic acid (AA)-functionalized antibody specifically recognized AFB1 and immobilized on the surface of the electrode. Finally, a large number of cyclopentenyl ferrocene (CFc) monomers were grafted onto the electrode surface under the action of the catalyst AquaMet, forming an electroactive long-chain polymer that results in a significant signal enhancement. Under optimal conditions, the detection limit of the method was 0.34 fg/mL with a detection range of 2 fg/mL-2 ng/mL. The proposed sensor is highly selective due to the high specificity of the aptamer and antibody to the target. In addition, the sensor has been successfully applied to detect AFB1 in real samples with recoveries ranging from 92.91% to 107.20%.

A dual-mechanism-driven electrochemiluminescence aptasensor for sensitive detection of β-amyloid peptides.

β-Amyloid (Aβ) peptides can bind both Cu2+ and heme cofactors simultaneously to form heme-Cu2+-Aβ complexes, which are proposed to generate toxic partially reduced oxygen species (PROS, e.g., H2O2) and play a vital role in Alzheimer's disease (AD). In this paper, a competitive dual-mechanism-driven electrochemiluminescence (ECL) aptasensor integrating the synergistic enhancement and steric hindrance effect was described for Aβ detection. Specifically, graphite carbon nitride (g-C3N4) as an effective ECL luminescent substrate and Au nanoparticles were sequentially assembled on the Au electrode surface, and then a thiol-modified aptamer for capturing Aβ peptide was attached to the surface of the electrode through the Au-S bond. Aβ peptides were simultaneously incubated with heme and Cu2+, and the forming heme-Cu2+-Aβ complexes were subsequently anchored on the electrode through the specific recognition between the target Aβ and the aptamer. When the concentration of the target Aβ is low, the synergistic enhancement effect arising from K2S2O8 with in situ generated H2O2 is predominant, resulting in an increase in the ECL signal of g-C3N4. In contrast, when the concentration of Aβ is high, the steric hindrance effect generated from heme-Cu2+-Aβ complexes is dominant, leading to a decrease in the ECL signal. The present sensor exhibits a favorable linear response for the detection of Aβ with a relatively low detection limit of 0.24 pM, and provides a more sensitive and selective platform for bioanalysis.

Detection of Glycated Albumin Using a Novel Field Effect Aptasensor

In this paper, we propose a new field effect (FE) sensor for fast detection of glycated albumin (GA) based on thiol modified aptamer (TMA). A p-type Si/ (100 nm) SiO2 wafer was used, where the SiO2 layer acts as the gate insulator. This gate insulator is then decorated with a layer of randomly oriented ZnO nanorods, coated with 40 nm of Au to act as binding sites for the TMA. We have shown that the addition of GA significantly reduces the conductivity of the p-type Si channel, which can be used to determine the GA concentration in the test solution. The ZnO/Au/TMA/GA layer can induce negative charges in the Si channel underneath the gate insulator, which is followed by the depletion of the positively charged carriers of the Si, decreasing the conductivity. Accordingly, the charge carriers in the Si channel show a steady decline with increasing GA concentration. We have shown that this decrease in conductivity can be further tuned by varying the gate voltage. The sensor presented here can be used as a fast and reliable sensor for determination of GA.

Highly sensitive detection for cocaine using an aptamer–cocaine–aptamer method

In this study, a thiol-modified aptamer was immobilized on a gold nanoparticle-modified molybdenum disulfide composite material (MoS2@AuNPs), which could remove the interference of physical adsorption and reduce false positive signals.

Label-free aptamer-based detection of microcystin-LR using a microcantilever array biosensor

Cyanotoxins, produced by cyanobacteria, are a series of widely present toxins frequently found in fresh water during algal blooms. They are extremely hazardous and persistent, which makes them a serious threat to human and animal health. In this paper, we developed an aptamer-based microcantilever array sensor to detect microcystin-leucine-arginine (MC-LR), one of the most concerned liver toxin. For this assay, an easily synthesized thiol-modified aptamer with specific recognition for MC-LR was used as a probe. The aptamer was covalently and directionally immobilized on the gold surface of a microcantilever by one-step immobilization via its thiol group, which simplified the conventional preparation of micocantilever array sensors. Interactions between the immobilized aptamer and MC-LR successfully changed the surface stress of the microcantilever, resulting in a bending conformation. The detection range was from 1 to 500 μg L−1, and the cantilever deflection had a good linear relationship within the concentration range of 1–50 μg L−1. Additionally, this sensor could identify MC-LR from other congeners. Thus, the aptamer-based microcantilever sensor operated in stress mode could achieve simple, rapid, real-time, label-free and quantitative detection of MC-LR, making it a convenient and economical approach for MC-LR detection. The aptamer-based microcantilever array sensor has great potential for detecting various cyanotoxins while new aptamers specific for cyanotoxins are available, which may be developed to monitor the environment and protect life health.

Selection of a new Mycobacterium tuberculosis H37Rv aptamer and its application in the construction of a SWCNT/aptamer/Au-IDE MSPQC H37Rv sensor

A rapid and accurate detection method for Mycobacterium tuberculosis (M. tuberculosis) is essential for effectively treating tuberculosis. However, current detection methods cannot meet these clinical requirements because the methods are slow or of low specificity. Consequently, a new highly specific ssDNA aptamer against M. tuberculosis reference strain H37Rv was selected by using the whole-cell systematic evolution of ligands by exponential enrichment technique. The selected aptamer was used to construct a fast and highly specific H37Rv sensor. The probe was produced by immobilizing thiol-modified aptamer on an Au interdigital electrode (Au-IDE) of a multichannel series piezoelectric quartz crystal (MSPQC) through Au-S bonding, and then single-walled carbon nanotubes (SWCNTs) were bonded on the aptamer by π-π stacking. SWCNTs were used as a signal indicator because of their considerable difference in conductivity compared with H37Rv. When H37Rv is present, it replaces the SWCNTs because it binds to the aptamer much more strongly than SWCNTs do. The replacement of SWCNTs by H37Rv resulted in a large change in the electrical properties, and this change was detected by the MSPQC. The proposed sensor is highly selective and can distinguish H37Rv from Mycobacterium smegmatis (M. smegmatis) and Bacillus Calmette-Guerin vaccine (BCG). The detection time was 70min and the detection limit was 100cfu/mL. Compared with conventional methods, this new SWCNT/aptamer/Au-IDE MSPQC H37Rv sensor was specific, rapid, and sensitive, and it holds great potential for the early detection of H37Rv in clinical diagnosis.

A fluorescence aptasensor based on semiconductor quantum dots and MoS2 nanosheets for ochratoxin A detection

Molybdenum disulfide (MoS2) nanosheet has been extensively investigated as an effective fluorescence quencher. However, its combination with semiconductor quantum dots (QDs) for the development of an aptasensor based on the fluorescence quenching has not be explored. Herein, a novel aptamer-CdTe QDs-MoS2 nanosheets fluorescence quenching system was developed for the detection of ochratoxin A (OTA) with QDs as donors and MoS2 nanosheets as quenchers. The QDs-conjugated aptamers with the linear conformation can assemble on the MoS2 nanosheets, causing the fluorescence quenching. In the presence of OTA, the aptamers wrap around the targets to form folded structures with a low affinity to the nanosheets, thus resulting in an OTA concentration-dependent restoration of the fluorescence intensity. The platform shows good specificity, a limit of detection of 1.0ng/mL and a dynamic range over 1.0–1000ng/mL in laboratory buffers. The platform is also successfully used to detect OTA in spiked red wine samples, verifying its feasibility in real sample analysis. The QDs-MoS2 nanosheets fluorescence quenching system could be easily adapted to measure other biomolecules by changing the thiol-modified aptamer.

Selection and Characterization of DNA Aptamers for Electrochemical Biosensing of Carbendazim.

This article reports a novel aptamer-based impedimetric detection of carbendazim, a commonly used benzimidazole fungicide in agriculture. High affinity and specificity DNA aptamers against carbendazim were successfully selected using systematic evolution of ligand by exponential enrichment (SELEX). The dissociation constants (Kds) of the selected DNA aptamers after 10 in vitro selection cycles were characterized using fluorescence-based assays showing values in the nanomolar range. The aptamer which showed the highest degree of affinity and conformation change was used to fabricate an electrochemical aptasensor via self-assembly of thiol-modified aptamer on gold electrodes. The aptasensor exploits the specific recognition of carbendazim by the aptamer immobilized on the gold surface which leads to conformational changes in the aptamer structure. This conformational change alters the access of a ferrocyanide/ferricyanide redox couple to the aptasensor surface. The aptasensor response is thus measured by following the increase in the electron transfer resistance of the redox couple using Faradaic electrochemical impedance spectroscopy. This method allowed a selective and sensitive label-free detection of carbendazim within a range of 10 pg/mL-10 ng/mL with a limit of detection of 8.2 pg/mL. The aptasensor did not show cross reactivity with other commonly used pesticides such as fenamiphos, isoproturon, atrazine, linuron, thiamethoxam, trifluralin, carbaryl, and methyl parathion. Moreover, the aptasensor has been applied in different spiked food matrixes showing high recovery percentages. We believe that the proposed aptasensor is a promising alternative to the currently used methods for carbendazim monitoring.

Electrochemiluminescence aptasensor of TiO2/CdS:Mn hybrids for ultrasensitive detection of cytochrome c

A novel electrochemiluminescence (ECL) aptasensor was proposed for ultrasensitive detection of cytochrome c (cyt c) using CdS:Mn quantum dot-modified TiO2 nanowires (NWs) as electrode. The Mn-doped CdS was deposited on the TiO2 NWs by successive ion layer adsorption and reaction (SILAR) as ECL emitter, on which thiol-modified aptamer of cyt c was attached via Cd–S bond. Due to the high photo-electrical transfer efficiency, the as-prepared aptasensor shows high selectivity and sensitivity towards cyt c with a detection limit of 9.5fM and a linear range from 50fM to 125pM.

Near-Surface Oxidized Sulfur Modifications and Self-Assembly of Thiol-Modified Aptamer on Au Thin Film Substrates Influenced by Piranha Treatment

Self-assembly of thiol-modified oligonucleotides on Au films has great importance for biosensor applications. Prior to the self-assembly, a piranha treatment (PT) is commonly used to clean the Au surface. Here we report that near-surface oxidized sulfur modifications on Au thin films by PT for longer than 60 s have serious effects on the self-assembled monolayer (SAM) formation of thiol-modified single-stranded thrombin binding aptamer (s-TBA), and a PT time of 10-30 s is optimal for s-TBA SAM formation. These results have important implication to SAM formation of biomolecules, especially for the thiol-modified ones where a careful consideration of this key step could significantly enhance the SAM formation and biosensor performance.