Aptamer-based Detection Research Articles

Morphology controlled NiO nanostructures as fluorescent quenchers for highly sensitive aptamer-based FRET detection of ochratoxin A

Herein, we presented a comprehensive investigation of structural properties of nickel oxide (NiO) nanomaterials prepared by rationally designed temperature-controlled synthesis. A series of eight types (A-H) of nickel oxide nanomaterials were synthesized by hydrothermal approach at different calcination temperatures (40–180) °C. Depending on temperature, different sizes and shapes were obtained including spherical nanoparticles, nanoclusters and flower-like morphologies, as characterized by X-ray diffraction (XRD), Scanning electron microscopy (SEM) and Raman spectroscopy. The best fluorescence quenching properties were obtained with spherical NiO-B nanoparticles 18 nm, prepared at 60 °C. The optimized NiO was used to develop an apta-assay for the detection of a food-relevant toxin, ochratoxin-A (OTA). In the presence of OTA, the biomolecular binding induced changes in the FAM labelled-aptamer structure and preferential formation of an aptamer-OTA complex. The method exhibited a limit of detection of 0.49 ng.L−1 with a wide linear range from 0.5 to 200 ng.L−1. The results demonstrate not only that rationally designed NiO nanostructures of varying shapes and sizes can be obtained by temperature-controlled synthesis, but they are also powerful fluorescent quenchers.

Aptamer-Based Detection of Circulating Targets for Precision Medicine.

The past decade has witnessed ongoing progress in precision medicine to improve human health. As an emerging diagnostic technique, liquid biopsy can provide real-time, comprehensive, dynamic physiological and pathological information in a noninvasive manner, opening a new window for precision medicine. Liquid biopsy depends on the sensitive and reliable detection of circulating targets (e.g., cells, extracellular vesicles, proteins, microRNAs) from body fluids, the performance of which is largely governed by recognition ligands. Aptamers are single-stranded functional oligonucleotides, capable of folding into unique tertiary structures to bind to their targets with superior specificity and affinity. Their mature evolution procedure, facile modification, and affinity regulation, as well as versatile structural design and engineering, make aptamers ideal recognition ligands for liquid biopsy. In this review, we present a broad overview of aptamer-based liquid biopsy techniques for precision medicine. We begin with recent advances in aptamer selection, followed by a summary of state-of-the-art strategies for multivalent aptamer assembly and aptamer interface modification. We will further describe aptamer-based micro-/nanoisolation platforms, aptamer-enabled release methods, and aptamer-assisted signal amplification and detection strategies. Finally, we present our perspectives regarding the opportunities and challenges of aptamer-based liquid biopsy for precision medicine.

High selectivity sensing of bovine serum albumin: The combination of glass nanopore and molecularly imprinted technology

Recently, resistive-pulse based nanopore analysis has made great progress. However, when it is used for the detection of similar substances, such as proteins, DNA, RNA and other biological molecules, the signal is poor. In order to realize the purpose of good testing, the solutions proposed by researchers include surface modification of nanopore or special data processing equipment and software, which are still complicated. In this manuscript, molecularly imprinted technology and nanopore sensing technology are combined together, based on the specific rebinding of molecularly imprinted polymer coated SiO2 nanoparticles to bovine serum albumin, the nanoparticles at different imprinting stages can be distinguished through resistive-pulse signals when they pass through the nanopores, achieving high selective and sensitive detection of bovine serum albumin in complex samples. The linear range of this nanopore-based sensor is from 0.5 nmol/L to 50 nmol/L with a 0.3 nmol/L detection limit. This method also possesses the advantages of strong specificity, good selectivity of molecularly imprinted polymer for target recognition and adsorption, and the controllable diameter and easy preparation of solid nanopores. Compared with the antibody-based or aptamer-based protein detection strategy, method in this manuscript is simple and easy to implement, which not only provides good selectivity and sensitivity for the detection of proteins in complex biomedical samples, but also opens up new application fields for nanopore sensing.

Oligonucleotide aptamers for pathogen detection and infectious disease control.

During an epidemic or pandemic, the primary task is to rapidly develop precise diagnostic approaches and effective therapeutics. Oligonucleotide aptamer-based pathogen detection assays and control therapeutics are promising, as aptamers that specifically recognize and block pathogens can be quickly developed and produced through simple chemical synthesis. This work reviews common aptamer-based diagnostic techniques for communicable diseases and summarizes currently available aptamers that target various pathogens, including the SARS-CoV-2 virus. Moreover, this review discusses how oligonucleotide aptamers might be leveraged to control pathogen propagation and improve host immune system responses. This review offers a comprehensive data source to the further develop aptamer-based diagnostics and therapeutics specific for infectious diseases.

Target-induced activation of polymerase activity for recycling signal amplification cascades for sensitive aptamer-based detection of biomarkers.

It is of great importance to develop biosensing methods for the sensitive and selective analysis of biomarkers at very low levels in biological samples. Using a new target-induced activation of the DNA polymerase activity for recycling amplification cascades, we describe an aptamer-based method for highly sensitive detection of platelet-derived growth factor BB (PDGF-BB) in human serums. The polymerase activity is initially inhibited by the binding of the polymerase to the enzyme aptamer sequence. PDGF-BB associates with and switches a PDGF-BB binding aptamer to trigger the release of an active polymerase, which further initiates the simultaneous recycling of the target PDGF-BB molecules and the enzyme aptamer sequence for the subsequent displacement of the fluorescently quenched probes to recover the fluorescence. Due to two recycling cascades, substantial fluorescence magnification is obtained for the highly sensitive detection of PDGF-BB with a low detection limit of 5.1 pM. Moreover, the potential applicability of this method for real samples was verified by determining PDGF-BB in diluted human serums, relying on the excellent specificity and selectivity of the aptamer. The demonstration of the PDGF-BB assay method here thus can be expanded for the construction of diverse sensing platforms for detecting different trace biomarkers with the integration of an elaborate design of the aptamer probes.

Combining aptamers and antibodies: Lateral flow quantification for thrombin and interleukin-6 with smartphone readout

Graphical representation of the developed assay, showing positive and negative assay results. • Dual use of aptamer and antibodies in one lateral flow method. • Quantification of clinically relevant targets in plasma. • Precise and direct determination of QD-tags by smartphone-based images. Modern strategies in precision medicine require diagnostic tools for fast assessment of biomarkers. A popular and well-established assay format for the rapid detection of disease markers directly at the point of care is the lateral flow assay, which enables medical staff to directly use body fluids such as blood or urine for diagnosis, without the need for a professional laboratory environment. Interleukin-6 and thrombin are clinically relevant biomarkers that are associated with infectious diseases, inflammation, and blood coagulation, and can provide valuable information on the status and treatment responses of patients with COVID-19. This work presents a novel method for the quantification of these biomarkers by using fluorescent green and red quantum dots as labels for interleukin-6 antibodies and thrombin binding aptamers, respectively. For readout, a 3D printed smartphone imager with a built in UV-LED light source is used. Through separation of RGB-channels, the acquired images can be processed to achieve a fully functional duplex lateral flow assay for simultaneous quantification of interleukin-6 and thrombin on the same test line (optical multiplexing). Furthermore, the assay performs well in complex samples (10 % serum samples). In conclusion, this novel combination of antibody and aptamer-based detection in a single lateral flow assay reduces turnaround time, and the user-friendly smartphone imager facilitates availability, particularly in low resource settings.

Aptamer-based microcantilever-array biosensor for ultra-sensitive and rapid detection of okadaic acid

Okadaic acid (OA) is widely present in seafood and consuming OA contaminated seafood can cause carcinogenic or teratogenic effects. Therefore, developing ultra-sensitive and rapid detection methods for OA is crucial. Here, an aptamer-based microcantilever array sensor was developed for ultra-sensitive and rapid detection of OA. OA aptamers were immobilized on a microcantilever through a thiol group in one step, thus greatly simplifying cumbersome preprocessing procedures. The detection limit of this assay for OA is 1 pg mL−1, the lowest of reported aptamer-based OA detection method. More importantly, compared with reported methods, our method based on micro-cantilever array sensor has the advantages of being rapid, label-free, economical and more applicable for food sample detection. We proposed a method of using enzyme-catalyzed amplification on microcantilever arrays and then used this method to verify the successful modification of aptamers. The sensor also showed the applicability in food sample detection and specificity to OA in shellfish toxin. Therefore, our results prove that this highly sensitive assay based on microcantilever sensor has practical application prospect in assessment in the quality and toxicity monitoring of seafood.

Aptamer-Based Detection of Ampicillin in Urine Samples.

The misuse of antibiotics in health care has led to increasing levels of drug resistant infections (DRI’s) occurring in the general population. Most technologies developed for the detection of DRI’s typically focus on phenotyping or genotyping bacterial resistance rather than on the underlying cause and spread of DRI’s; namely the misuse of antibiotics. An aptameric based assay has been developed for the monitoring of ampicillin in urine samples, for use in determining optimal antibiotic dosage and monitoring patient compliance with treatment. The fluorescently labelled aptamers were shown to perform optimally at pH 7, ideal for buffered clinical urine samples, with limits of detection as low as 20.6 nM, allowing for determination of ampicillin in urine in the clinically relevant range of concentrations (100 nM to 100 µM). As the assay requires incubation for only 1 h with a small sample volume, 50 to 150 µL, the test would fit within current healthcare pathways, simplifying the adoption of the technology.

Advances in Exosome Analysis Methods with an Emphasis on Electrochemistry

Exosomes, small extracellular vesicles, are released by various cell types. They are found in bodily fluids, including blood, urine, serum, and saliva, and play essential roles in intercellular communication. Exosomes contain various biomarkers, such as nucleic acids and proteins, that reflect the status of their parent cells. Since they influence tumorigenesis and metastasis in cancer patients, exosomes are excellent noninvasive potential indicators for early cancer detection. Aptamers with specific binding properties have distinct advantages over antibodies, making them effective versatile bioreceptors for the detection of exosome biomarkers. Here, we review various aptamer-based exosome detection approaches based on signaling methods, such as fluorescence, colorimetry, and chemiluminescence, focusing on electrochemical strategies that are easier, cost-effective, and more sensitive than others. Further, we discuss the clinical applications of electrochemical exosome analysis strategies as well as future research directions in this field.

An aptamer-based magnetic flow cytometer using matched filtering

Facing unprecedented population-ageing, the management of noncommunicable diseases (NCDs) urgently needs a point-of-care (PoC) testing infrastructure. Magnetic flow cytometers are one such solution for rapid cancer cellular detection in a PoC setting. In this work, we report a giant magnetoresistive spin-valve (GMR SV) biosensor array with a multi-stripe sensor geometry and matched filtering to improve detection accuracy without compromising throughput. The carefully designed sensor geometry generates a characteristic signature when cells labeled with magnetic nanoparticles (MNPs) pass by thus enabling multi-parametric measurement like optical flow cytometers (FCMs). Enumeration and multi-parametric information were successfully measured across two decades of throughput (37 — 2730 cells/min). 10-μm polymer microspheres were used as a biomimetic model where MNPs and MNP-decorated polymer conjugates were flown over the GMR SV sensor array and detected with a signal-to-noise ratio (SNR) as low as 2.5 dB due to the processing gain afforded by the matched filtering. The performance was compared against optical observation, exhibiting a 92% detection efficiency. The system achieved a 95% counting accuracy for biomimetic models and 98% for aptamer-based pancreatic cancer cell detection. This system demonstrates the ability to perform reliable flow cytometry toward PoC diagnostics to benefit NCD control plans.

A self-calibrating electrochemical aptasensing platform: Correcting external interference errors for the reliable and stable detection of avian influenza viruses

Conventional electrochemical biosensing systems rely on a single output signal, which limits their certain practical application, specifically from the viewpoint of external interference factors causing electrochemical signal errors. This study reports a self-calibrating dual-electrode based electrochemical aptasensor for the reliable and independent detection of avian influenza viruses (AIVs), which are the primary cause of highly contagious respiratory diseases, under external interference factors. Both electrodes were fabricated using tungsten rods surface-modified with a 3D nanostructured porous silica film (3DNRE). Subsequently, methylene blue (MB) was loaded as a redox-active material into the pores and capped with corresponding aptamer. One electrode was capped with an anti-AIV nucleoprotein (NP) aptamer (AptAIV-MB@3DNRE) allowing target-specific binding, resulting in changes in electrochemical signal upon diffusional release of the loaded redox molecules. The other electrode was capped with a control aptamer (Aptcon-MB@3DNRE), serving as a reference to correct false responses generated by nonspecific aptamer detachment and MB release under environmental changes in pH and ion strength and presence of nontarget molecules from cell lysis debris. In the dual-electrode platform, Aptcon-MB@3DNRE provides a corrected baseline for the fluctuating original output signals from AptAIV-MB@3DNRE. Consequently, this dual-electrode platform exhibits excellent output-signal stability (relative standard deviation, RSD: 5.86%) compared to a conventional single-electrode platform (RSD: 30.13%) at equivalent concentrations of AIV NP samples under different reaction buffer conditions. Moreover, no further purification and washing steps were required, indicating that the strategy may represent a universal and reliable platform for the electrochemical aptamer-based detection of various biomolecules.

Selection of potential aptamers for specific growth stage detection of Yersinia enterocolitica.

Yersinia enterocolitica remains a threat to public health, and a sensitive detection method is a prerequisite due to its complicated diagnosis associated with slow growth. Recently, aptamer-based detection systems have played a vital role in the development of simple, rapid, sensitive, and specific detection methods. Herein, highly specific ssDNA aptamers were screened against Y. enterocolitica at the different growth stages by whole cell-SELEX. Cells at different growth stages were harvested and incubated with an ssDNA library to get an enriched pool of specific aptamer candidates. After the 10th round of SELEX, the enriched pool was sequenced and grouped into seven families based on homology and similarity of the secondary structure. Flow cytometry analysis revealed that the aptamers M1, M5, and M7 with Kd values of 37.93 ± 7.88 nM, 74.96 ± 21.34 nM, and 73.02 ± 18.76 nM had the highest affinity and specificity to the target, respectively. The selected aptamers showed binding to the different growth stages of Y. enterocolitica with a significant increase in the gated fluorescence. Our aptamer selection strategy is convenient, and the developed aptamer can be useful for an accurate and reliable detection system.

Polymerization nicking-triggered LAMP cascades enable exceptional signal amplification for aptamer-based label-free detection of trace proteins in human serum

Detecting molecular biomarkers in high sensitivity plays an important role in the diagnosis of various diseases at the early stage. Here, by combining the target-induced polymerization nicking reaction (TIPNR) with the loop-mediated isothermal amplification (LAMP), we describe an ultrasensitive and label-free aptamer-based sensing method for detecting low levels of proteins in human serum by using thrombin as the model target analyte. The target thrombin binds and causes spontaneous assembly of two distinct aptamer probes to form the templates for the polymerization nicking reaction recycling amplification to produce many forward inner primer sequences. Subsequently, downstream LAMP reactions are initiated by these sequences for the generation of tremendous DNA hairpins with various lengths via automated cyclic strand displacement reactions. The SYBR Green I organic dye further binds the many hairpins to show drastically amplified fluorescence for ultrasensitive detection of thrombin down to 3.6 fM in the linear range from 0.01 pM to 10 nM. Such a sensing method based on aptamers has high discrimination capability for the target molecules against other non-specific proteins and is applicable for diluted serum samples. With the successful demonstration of the substantial signal amplification ability and simplicity feature of this assay approach, highly sensitive and convenient detection of other disease biomarkers with this method can be envisioned in the near future.

Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers

Infection of various pathogenic bacteria causes severe illness to human beings. Despite the research advances, current identification tools still exhibit limitations in detecting Gram-negative bacteria with high accuracy. In this study, we isolated single-stranded DNA aptamers against multiple Gram-negative bacterial species using Toggle-cell-SELEX (systemic evolution of ligands by exponential enrichment) and constructed an aptamer-based detection tool towards bacterial secretory cargo released from outer membranes of Gram-negative bacteria. Three Gram-negative bacteria, Escherichia coli DH5α, E. coli K12, and Serratia marcescens, were sequentially incubated with the pool of random DNA sequences at each SELEX loop. Two aptamers selected, GN6 and GN12, were 4.2-times and 3.6-times higher binding to 108 cells of Gram-negative bacteria than to Gram-positive bacteria tested, respectively. Using GN6 aptamer, we constructed an Enzyme-linked aptamer assay (ELAA) to detect bacterial outer membrane vesicles (OMVs) of Gram-negative bacteria, which contain several outer membrane proteins with potent immunostimulatory effects. The GN6-ELAA showed high sensitivity to detect as low as 25 ng/mL bacterial OMVs. Aptamers developed in this study show a great potential to facilitate medical diagnosis and early detection of bacterial terrorism, based on the ability to detect bacterial OMVs of multiple Gram-negative bacteria.

Two-Step Energy Transfer Dynamics in Conjugated Polymer and Dye-Labeled Aptamer-Based Potassium Ion Detection Assay.

We recently implemented highly sensitive detection systems for photo-sensitizing potassium ions (K+) based on two-step Förster resonance energy transfer (FRET). As a successive study for quantitative understanding of energy transfer processes in terms of the exciton population, we investigated the fluorescence decay dynamics in conjugated polymers and an aptamer-based 6-carboxyfluorescein (6-FAM)/6-carboxytetramethylrhodamine (TAMRA) complex. In the presence of K+ ions, the Guanine-rich aptamer enabled efficient two-step resonance energy transfer from conjugated polymers to dyed pairs of 6-FAM and TAMRA through the G-quadruplex phase. Although the fluorescence decay time of TAMRA barely changed, the fluorescence intensity was significantly increased. We also found that 6-FAM showed a decreased exciton population due the compensation of energy transfer to TAMRA by FRET from conjugated polymers, but a fluorescence quenching also occurred concomitantly. Consequently, the fluorescence intensity of TAMRA showed a 4-fold enhancement, where the initial transfer efficiency (~300%) rapidly saturated within ~0.5 ns and the plateau of transfer efficiency (~230%) remained afterward.

Application of Aptamers in Virus Detection and Antiviral Therapy.

Viral infections can cause serious diseases for humans and animals. Accurate and early detection of viruses is often crucial for clinical diagnosis and therapy. Aptamers are mostly single-stranded nucleotide sequences that are artificially synthesized by an in vitro technology known as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Similar to antibodies, aptamers bind specifically to their targets. However, compared with antibody, aptamers are easy to synthesize and modify and can bind to a broad range of targets. Thus, aptamers are promising for detecting viruses and treating viral infections. In this review, we briefly introduce aptamer-based biosensors (aptasensors) and describe their applications in rapid detection of viruses and as antiviral agents in treating infections. We summarize available data about the use of aptamers to detect and inhibit viruses. Furthermore, for the first time, we list aptamers specific to different viruses that have been screened out but have not yet been used for detecting viruses or treating viral infections. Finally, we analyze barriers and developing perspectives in the application of aptamer-based virus detection and therapeutics.

Photoelectrochemical aptasensing of ofloxacin based on the use of a TiO2 nanotube array co-sensitized with a nanocomposite prepared from polydopamine and Ag2S nanoparticles.

A photoelectrochemical (PEC) method is described for aptamer-based detection of ofloxacin (OFL). It is making use of a TiO2 nanotube array (NTA) that is sensitized with a structure composed of polydopamine and silver sulfide nanoparticles. The NTA were prepared by a two-step synthetic method. First, the TiO2 nanotube electrode was covered with Ag2S nanoparticles via successive ionic layer adsorption and reaction strategy. Next, they were coated with a thin film of polydopamine (PDA) by in-situ polymerization. The inorganic/organic nanocomposites exhibit distinctly enhanced visible-light PEC activity. This was exploited to fabricate a PEC aptasensor. The PDA film serves as both the sensitizer for charge separation and as a support to bind the aptamer against OFL. The aptasensor undergoes a decrease in photocurrent due to the formation of the aptamer-OFL complex. Under the optimized conditions and at a typical working potential of 0V (vs. Hg/Hg2Cl2), the NTA has a linear response in the 5.0 pM to 100nM OFL concentration range and a 0.75 pM detection limit (at S/N = 3). The aptasensor was successfully applied to the determination of OFL in spiked milk samples. Graphical abstract Schematic illustration for the preparation and mechanism of the photoelectrochemical aptasensor for ofloxacin. TiO2 NTs: TiO2 nanotube arrays; PDA: polydopamine; MCH: 6-mercapto-1-hexanol; OFL: ofloxacin; PEC: photoelectrochemistry; CB: conduction band; VB: valence band; LUMO: the lowest unoccupied molecular orbital; HOMO: the highest occupied molecular orbital; AA: ascorbic acid.

Inkjet printed electrochemical aptasensor for detection of Hg2+ in organic solvents

Real-time detection of heavy metal traces from industrial waste is key to control any potential environmental pollution by early critical point intervention. This opens a market niche for real-time, portable, and cost-efficient devices for environmental and biosafety monitoring. Despite the rapid growth of heavy metal monitoring as a research field, and the promising performance of an increasing number of novel platforms reported, the chances to transfer the lab-scale platforms developed to successful commercialization stages are dramatically low.This work proposes for the first time the use of ink-jet printed gold electrodes as a reliable, stable and fully-scalable approach for aptamer-based detection of mercury (Hg2+) traces in water and organic solvents. A controlled fabrication process was demonstrated by keeping the 90% of the charge transfer (RCT) values obtained from the as-fabricated electrodes (n = 11) within ± 1 SD (RSD = 2.92%) from the mean value and following a mesokurtic normal-like distribution (p = 0.905). The as-fabricated platform achieved a LOD of 0.01 ppm in water and enhanced sensitivity of 2-fold when tested in organic solvents, with a LOD of 0.005 ppm which is far below the minimal sensitive required for practical implementation, according to the EPA stipulations. Finally, the aptamer-based gold ink-jet printed platform provided with enough evidence to state that the signal collected from Hg2+ was significantly different (p < 0.001) from the signal provided by interferent ions tested (arsenic, lead and cadmium) at the same concentration (50 ppm), evidencing a high specificity. From the best of our knowledge, this work reports for the first time a gold inkjet-printed impedimetric platform for aptamer-based detection of Hg2+ in organic solvents, showing promising viability for future prototyping and scaling-up.

Highly Sensitive Detection of Protein Biomarkers Using Graphene Field-Effect Transistor Integrated with a Microfluidic Platform

In this work, we present a highly sensitive biosensing platform for the detection of protein biomarkers by utilizing aptamer-modified graphene field-effect transistors (GFETs) contained within a microfluidic chip. We have chosen thrombin as a model biomarker and its corresponding DNA-based aptamer as a target receptor which is anchored to the graphene surface to modulate the drain current of the GFET. The proposed biosensing platform has the potential to be used in early-stage disease diagnosis and in applications related to lab-on-a-chip-based health monitoring technology. Aptamer-based biosensing offers many advantages including low-cost, chemical and thermal stability, mass producibility, long shelf-life and reusability. Among different target analytes, protein biomarker detection is critical in early screening and diagnostics of diseases. Thrombin is an important protein biomarker which is produced by the body and plays a significant role in blood clotting or coagulation process or regulation of tumor growth [1]. Therefore, detection of thrombin is key to monitoring a number of cardiovascular diseases. Graphene-based FET (GFET) has become a highly promising candidate as next-generation biosensors due to the various inherent advantages it offers such as facile integration with the existing planar technology due to its 2-dimensional structure, label-free sensing, and high sensitivity. However, one of the major challenges with GFET-based biosensing is the lack of reproducibility due to each graphene layer having different physical dimensions and therefore electronic properties. Moreover, since GFET is highly sensitive to the charge disturbances at the FET channel, the sample liquid that is in contact with the graphene surface must be controlled. Therefore, integration of GFET biosensor with a microfluidic device is expected to enhance the sensing performances in terms of sensitivity and reproducibility. In this work, we have implemented a microfluidic chip integrated with GFET device to build a miniaturized platform for aptamer-based detection of a protein biomarker. The GFET devices were fabricated by transferring CVD (chemical vapor deposition) grown graphene onto the microfabricated gold electrodes on the Si/SiO2substrate where the electrodes form the source and the drainas schematically depicted in Figure 1. The FET devices were then sealed with a PDMS block containing a microfluidic channel as shown in Figure 2. The graphene channel was then functionalized with amine reactive 1-pyrene butyric acid N-hydroxysuccinimide ester (PBASE) by delivering the PBASE dissolved in dimethyl formamide through the microfluidic channel. The PBASE was immobilized on graphene surface via the π-π stacking interaction between graphene and the pyrene group of PBASE. The graphene channel was then further functionalized with the aptamers by flowing the amine terminated thrombin-binding aptamer solution through the microfluidic chip. The aptamers were crosslinked with the PBASE on graphene surface via the covalent linkage between the amine-reactive succinimide group of PBASE and the amine group of the aptamers [2]. For the biomarker detection, various concentrations of thrombin solutions were introduced to the GFET device through the microfluidic flow system. For a given concentration of thrombin, the drain current (IDS) vs. gate voltage (VGS) characteristics were measured in order to observe the shift in the Dirac voltage (VDirac). Figure 3 shows the Raman spectra of graphene on Si/SiO2 substrate before and after PBASE functionalization. The additional peak at 1618 cm-1 in Figure 3(a) represents the pyrene group resonance peak due to π – π stacking interaction [3] between the aromatic pyrenyl group of PBASE and the basal plane of graphene, confirming the immobilization of PBASE on graphene. Figure 4 shows the UV-vis spectra of the device sample before and after the aptamer immobilization. The peak at 260 nm indicates the presence of aptamers on the device surface. The resulting measurements are presented in Figure 5. It can be seen that introduction of thrombin causes the Dirac voltage to shift to the right suggesting p-doping of the graphene surface [4]. This is consistent with the cationic nature of thrombin at neutral pH. The corresponding calibration curve is shown in Figure 6. The sensor shows a linear behavior over a wide range of concentrations from 1 nM to 1µM.

Split aptamer-based detection of adenosine triphosphate using surface enhanced Raman spectroscopy and two kinds of gold nanoparticles.

An ultrasensitive and highly selective method is described for the determination of adenosine triphosphate (ATP) via surface-enhanced Raman scattering (SERS). Two split aptamers are used for specific recognition of ATP. They were attached to two SERS substrates. The first was placed on a nanolayer of gold nanoparticle-decorated graphene oxide (GO/Au3), and the other on gold nanoparticles (Au2). When ATP is introduced, it will interact with the split aptamers on the gold nanostructures to form a sandwich structure that brings the GO/Au3 nanolayer and the Au2 nanoparticle in close proximity. Consequently, the SERS signal, best measured at 1072cm-1, is strongly enhanced. The sandwich structure also displays good water solubility and stability. Under optimized conditions, the SERS signal increases in the 10 pM-10nM ATP concentration range, and the limit of detection (LOD) is 0.85 pM. The method was applied to the determination of ATP in spiked human serum, and the LODs in serum and buffer are comparable. In our perception, the method has a wide scope in that numerous other aptamers may be used. This may result in a variety of other highly sensitive aptasensors for use in in-vitro diagnostics. Graphical abstract Schematic presentation of a self-assembly sandwich nanostructure as unique SERS assay platform for the sensitive detection of ATP.