We developed an efficient and fast method based on a protein microarray integrated with a microfluidic chip for the process of SELEX (systematic evolution of ligands by exponential enrichment). Lactoferrin from bovine milk was used as a target protein, while bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin and casein were used as negative proteins. They were separately dotted and immobilized to prepare the protein microarray and the resulting microarray was further integrated into a microfluidic chip for the SELEX (PMM–SELEX) process. The interaction between aptamer candidates and targets could be monitored using a fluorescence microarray scanner and the whole PMM–SELEX process was performed through seven-round selection. As a result, five aptamers (Lac-14, Lac-6a, Lac-9, Lac-5, Lac-3a) with high specificity and affinity can be repeatedly obtained during three times of independent repeated selection. Surface plasmon resonance (SPR) was used to calculate the dissociation constants (Kd). The aptamer Lac-6a was then used for detection of lactoferrin by fluorescence polarization. A linear response was observed for lactoferrin concentrations in the range of 0.78–50 μg mL−1 and the detection limit was 0.39 μg mL−1. Thus, the innovative PMM–SELEX presented shows stability, accuracy and high efficiency for aptamer screening.

Detection of rare circulating tumor cells (CTCs) in peripheral blood is a challenging, but necessary, task in order to diagnose early onset of metastatic cancer and to monitor treatment efficacy. Over the last decade, step-up produced aptamers have attracted great attention in clinical diagnosis. They have offered great promise for a broader range of cell-specific recognition and isolation. In particular, aptamer-functionalized magnetic particles for selective extraction of target CTCs have shown reduced damage to cells and relatively simple operation. Also, efforts to develop aptamer-functionalized microchannel/microstructures able to efficiently isolate target CTCs are continuing, and these efforts have brought more advanced geometrically designed substrates. Various aptamer-mediated cell release techniques are being developed to enable subsequent biological studies. This article reviews some of these advances in aptamer-functionalized nano/micro-materials for CTCs isolation and methods for releasing captured CTCs from aptamer-functionalized surfaces. Biological studies of CTCs after release are also discussed.

•A rapid visual protein detection method was developed for 96-well microplate arrays by using aptamer modifying silver nanoparticles.•An internal standard curve method based on immobilized SA proteins could be developed for quantitative detection of IgE.•The apt-AgNPs system showed a wider linear range and good specificity in human serum.A rapid, visual protein detection method of human immunoglobulin E (hIgE) was developed for 96-well microplate arrays by using aptamer modifying silver nanoparticles. An aptamer specifically recognizing IgE and biotinylated oligonucleotides were used to modify AgNPs and the resulting nanoprobes were employed to assay model analyte hIgE. When addition of the analyte as well as the probes to the microplate array on which Goat anti-human IgE was first immobilized, the signal of this “sandwich” format was further amplified by silver enhancement technique based on silver ions reductive reagents and achieved a visual dots in the microplates. To achieve quantitative analysis, a series of gradient concentration streptavidin (SA) was immobilized in the same well. As a result, an internal standard curve was plotted by the signal of different concentration of SA based on streptavidin-biotin coupling reaction. With the help of this curve, a quantitative detection of IgE was established and a detection limit of 20 ng mL−1 could be obtained. The presented method was further carried out for detection of human serum samples and relative consistent results were found.Figure optionsDownload full-size imageDownload as PowerPoint slide

•Two adjustable FRET systems are fabricated on the surface of Ag10NPs.•The nanosensor Ag-Alexa/Cy3/BHQ-2 was highly sensitive than that of Ag-Alexa/Cy3 and Alexa/Cy3/BHQ-2•Ag10NPs could increase the sensitivity of FERT sensor Alexa/Cy3/BHQ-2.•BHQ-2 could increase the sensitivity of FRET sensor Ag-Alexa/Cy3.We developed a silver decahedral nanoparticles (Ag10NPs)-enhanced ratiometric Fluorescence Resonance Energy Transfer (FRET) nanosensor based on two adjustable FRET modes. Alexa Fluor 488 (Alexa) and Cyanine3 (Cy3)-aptamer-Black hole quencher-2 (BHQ-2) were bound with Ag10NPs to form the ratiometric FRET nanosensor (Ag-Alexa/Cy3/BHQ-2). Alexa act as donor and Cy3 as acceptor in the FRET mode 1 while Cy3 was donor and BHQ-2 was acceptor in the FRET mode 2. In the absence of platelet-derived growth factor (PDGF-BB), the fluorescence intensity of Alexa was lowest while that of Cy3 was highest. Upon the addition of PDGF-BB, Cy3-aptamer-BHQ-2 binds with PDGF-BB resulting in the change of structure of aptamer. The fluorescence intensity of Alexa increased while that of Cy3 decreased. In addition, the fluorescence intensity ratio of Alexa to Cy3 increased remarkably with PDGF-BB concentration in the range of 0.4–400 ng/mL. A good linear response was obtained when the PDGF-BB concentrations were in the range of 3.1–200 ng/mL, with the limit of detection at 0.4 ng/mL. When compared to sensors without Ag10NPs (Alexa/Cy3/BHQ-2) and one without BHQ-2 (Ag-Alexa/Cy3), the new nanosensor Ag-Alexa/Cy3/BHQ-2 showed remarkable increase in sensitivity.

•MoS2/Ag nanohybrid was applied as a novel matrix in negative-ion MALDI-TOF MS.•The MoS2/Ag nanohybrid exerted synergistic effect on the detection of small molecules.•The MoS2/Ag nanohybrid showed good signal reproducibility and low background interferences comparing to organic matrices.•MoS2/Ag allows simultaneous analysis of multiple drugs and quantification of acetylsalicylic acid in spiked serum samples.This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS2/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS2. Moreover, both Ag nanoparticles and the edge of the MoS2 layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS2/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS2/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.

•The dual aptamers substitute for antibodies.•The silver-based microarray can enhance the sensitivity of proteins detection.•The silver-based microarray can detect multiplex proteins simultaneously.•The aptamer-based sandwich assay is sensitive and specific.In this work, aptamers-modified silver nanoparticles (AgNPs) were prepared as capture substrate, and fluorescent dyes-modified aptamers were synthesized as detection probes. The sandwich assay was based on dual aptamers, which was aimed to accomplish the highly sensitive detection of single protein and multiplex detection of proteins on one-spot. We found that aptamers-modified AgNPs based microarray was much superior to the aptamer based microarray in fluorescence detection of proteins. The result shows that the detection limit of the sandwich assay using AgNPs probes for thrombin or platelet-derived growth factor-BB (PDGF-BB) is 80 or 8 times lower than that of aptamers used directly. For multiplex detection of proteins, the detection limit was 625 pM for PDGF-BB and 21 pM for thrombin respectively. The sandwich assay based on dual aptamers and AgNPs was sensitive and specific.

•A novel and enzyme-free electrochemical aptasensor based on magnetic separation platform was designed.•Catalytic activity of silver nanoparticles towards the reduction of o-nitrophenol to o-aminophenol by NaBH4.•Direct electric readout from the products of electrodeposition of o-aminophenol and multiwalled carbon nanotubes.•Signal amplification by silver nanoparticles aggregates inducing by DNA hybridization.A novel and enzyme-free electrochemical aptasensor based on magnetic separation platform was designed for highly sensitive determination of PDGF-BB by virtue of utilizing catalytic activity of silver nanoparticles (AgNPs) towards the reduction of o-nitrophenol (o-NP) to o-aminophenol (o-AP) by NaBH4, and the direct electric readout from the product of electrodeposition of o-AP and multiwalled carbon nanotubes (MWCNTs) on screen printed electrode (SPE). Aptamers functionalized magnetic beads (apt-MBs) were used as capture probe, while AgNPs modified with aptamers and hybrid probes (apt-AgNPs-A and apt-AgNPs-B) were used as tags. In the presence of PDGF-BB, the apt-MBs and the AgNPs aggregates which were induced by in-situ hybridization of apt-AgNPs-A and apt-AgNPs-B formed a sandwich-like complex, followed by adding of NaBH4 and o-NP. Owing to the amplified catalysis of AgNPs aggregates conjugated in the complex, the o-NP was reduced by NaBH4 to o-AP, which was electro-co-deposition with MWCNTs on the SPE, producing a conducting nano-composite. The signal readout in this aptasensor for PDGF-BB was obtained by direct measurement of the poly(o-AP-MWCNTs) film on the SPE. Using this signal transduction protocol, the analytical performance of the electrochemical aptasensor was studied in detail. Under the optimal conditions, the assay displayed a wide dynamic working range of between 0.1 ng mL− 1 and 100 ng mL− 1with a detection limit of 60 pg mL− 1 relative to target PDGF-BB. Incorporation of good catalytic ability of hybridization inducing AgNPs aggregates and excellent electrochemical property of poly(o-AP-MWCNTs) nanocomposite could result in the amplification of the electrochemical signal, owing to the high loading of catalytic AgNPs and electro-co-deposition of MWCNTs. This method would have a promising future for application in clinical diagnostics.

•A colorimetric microfluidic chip-based silver nanoparticles aptasensor was designed.•The detection principle was based on the color of AgNPs without signal amplification.•This system combined microfluidic chip with colorimetric quantitative analysis.•The detection limit of thrombin was as low as 20 pM with excellent specificity.In this paper, a colorimetric silver nanoparticles aptasensor (aptamer-AgNPs) was developed for simple and straightforward detection of protein in microfluidic chip. Surface-functionalized microfluidic channels were employed as the capture platform. Then the mixture of target protein and aptamer-AgNPs were injected into the microfluidic channels for colorimetric detection. To demonstrate the performance of this detection platform, thrombin was chosen as a model target protein. Introduction of thrombin could form a sandwich-type complex involving immobilized AgNPs. The amount of aptamer-AgNPs on the complex augmented along with the increase of the thrombin concentration causing different color change that can be analyzed both by naked eyes and a flatbed scanner. This method is featured with low sample consumption, simple processes of microfluidic platform and straightforward colorimetric detection with aptamer-AgNPs. Thrombin at concentrations as low as 20 pM can be detected using this aptasensor without signal amplification. This work demonstrated that it had good selectivity over other proteins and it could be a useful strategy to detect other targets with two affinity binding sites for ligands as well.

We report on a silver decahedral nanoparticles (Ag10NPs)-based FRET (fluorescence resonance energy transfer) sensor for target cell imaging. Fluorophores-functionalized aptamers (Sgc8-FITC) were bound with Ag10NPs via the SH group on the aptamer to form Ag10-Sgc8-FITC. Then, quencher-carrying strands (BHQ-1) were hybridized with Sgc8-FITC to form a Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q). The sensor interacted with membrane protein tyrosine kinase-7 (PTK-7) on the CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. It was found that Ag10NPs largely enhanced the fluorescence intensity of FITC. The results also showed that the Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q) was not only superior to the bare FRET sensor (Sgc8-F/Q) and sensor Ag-Sgc8-F/Q but also highly sensitive and specific for CCRF-CEM cells imaging.

We fabricated a multifunctional theragnostic agent Ag-Sgc8-FAM for apoptosis-based cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, aptamers Sgc8 and TDO5 acted as recognizing molecules to bind CCRF-CEM and Ramos cells specifically. It was found that aptamer–silver conjugates (Ag-Sgc8, Ag-TDO5) could be internalized into cells by receptor-mediated endocytosis, inducing specific apoptosis of CCRF-CEM and Ramos cells. The apoptosis of cells depended on the concentration of aptamer–silver conjugates, as well as the incubation time between cells and aptamer-silver conjugates. The apoptotic effects on CCRF-CEM and Ramos cells were different. Annexin V/PI staining, AO/PI staining, MTT assays and ROS (reactive oxygen species) detection demonstrated the specific apoptosis of CCRF-CEM and Ramos cells. For fluorescence-enhanced cell imaging, Ag-Sgc8-FAM was prepared. Compared to Sgc8-FAM molecules, Ag-Sgc8-FAM was an excellent imaging agent as numerous Sgc8-FAM molecules were enriched on the surface of AgNPs for multiple binding with CCRF-CEM cells and signal amplification. Moreover, AgNPs could increase the fluorescence intensity of FAM by metal-enhanced fluorescence (MEF) effect. Therefore, aptamer–silver conjugates can be potential theragnostic agents for inducing specific apoptosis of cells and achieving cells imaging in real time.

A nanocomplex was developed for molecular sensing in living cells, based on the fluorophore-labeled aptamer and the polydopamine nanospheres (PDANS). Due to the interaction between ssDNA and PDANS, the aptamer was adsorbed onto the surface of PDANS forming the aptamer/PDANS nanocomplex, and the fluorescence was quenched by PDANS through Förster resonance energy transfer (FRET). In vitro assay, the introduction of adenosine triphosphate (ATP) led to the dissociation of the aptamer from the PDANS and the recovery of the fluorescence. The retained fluorescence of the nanocomplex was found to be linear with the concentration of ATP in the range of 0.01–2 mM, and the nanocomplex was highly selective toward ATP. For the strong protecting capability to nucleic acids from enzymatic cleavage and the excellent biocompatibility of PDANS, the nanocomplex was transported into cells and successfully realized “signal on” sensing of ATP in living cells; moreover, the nanocomplex could be employed for ATP semiquantification. This design provides a strategy to develop biosensors based on the polydopamine nanomaterials for intracellular molecules analysis. For the advantages of polydopamine, it would be an excellent candidate for many biological applications, such as gene and drug delivery, intracellular imaging, and in vivo monitoring.

•A fluorescence turn-on thrombin aptasensor using polyA-based AuNP probe is presented.•Utility of polyA-based nanoconjugates for protein bioassays is first demonstrated.•A divide-and-conquer strategy was applied to destroy the strong AuNP-polyA interaction.•I− and S2O32- were first adopted to detach the polyA-tailed DNA strands from AuNP surface.•Fluorescence turn-on bioassay was achieved using iodide-induced ligand displacement.Depending on the strong affinity of polyA sequence to gold (or silver) surface, applicability of polyA-tailed DNA–gold (or silver) nanoparticle conjugates in homogeneous and heterogeneous protein assays was first demonstrated. Interestingly, when using polyA-tailed, fluophore-labeled DNA–AuNP conjugate, it was found that iodide and thiosulfate anions could act as the ligand displacing reagent to detach polyA-tailed DNA strands from AuNP surface and simultaneously activate the AuNP-quenched fluorophores by destroying the polyA–AuNP interaction via a divide-and-conquer strategy. Based on this new discovery, we have developed a novel, cost-effective and sandwich-type fluorescence turn-on aptasensor for highly sensitive and specific thrombin detection, what took advantage of aptamer-conjugated magnetic beads (apt-MBs) for protein capture and separation, and iodide-induced fluorescence recovery of activatable polyA-based AuNP probes through ligand displacement for fluorescence turn-on detection. This proposed aptasensor could detect thrombin specifically with a detection limit as low as 89 pM, which was better than or comparable to many existing fluorescent thrombin assays. Importantly, employment of such polyA-based AuNP conjugate not only avoids the use of thiolated oligonucleotides and thiol-containing displacing reagents, but also offers new possibilities for fabricating convenient and cost-effective bioanalytical applications.

•A fluorescent sensing platform based on PDANS and Exo III was developed.•For Exo III-assisted target recycling, the signal for DNA detection was amplified.•An aptamer biosensor based on the PDANS was constructed for the assay of ATP.•The LOD of DNA and ATP was lower than other nanoquenchers-based biosensors.•The assay was fast, which could be completed in 45 minutes.Rapid, cost-effective, sensitive and specific analysis of biomolecules is important in the modern healthcare system. Here, a fluorescent biosensing platform based on the polydopamine nanospheres (PDANS) intergrating with Exonuclease III (Exo III) was developed. Due to the interaction between the ssDNA and the PDANS, the fluorescence of 6-carboxyfluorescein (FAM) labelled in the probe would been quenched by PDANS through FRET. While, in the present of the target DNA, the probe DNA would hybridize with the target DNA to form the double-strand DNA complex. Thus, Exo III could catalyze the stepwise removal of mononucleotides from 3′-terminus in the probe DNA, releasing the target DNA. As the FAM was released from the probe DNA, the fluorescence would no longer been quenched, led to the signal on. As one target DNA molecule could undergo a number of cycles to trigger the degradation of abundant probe DNA, Exo III-assisted target recycling would led to the amplification of the signal. The detection limit for DNA was 5 pM, which was 20 times lower than that without Exo III. And the assay time was largely shortened due to the faster signal recovery kinetics. What is more, this target recycling strategy was also applied to conduct an aptamer-based biosensing platform. The fluorescence intensity was also enhanced for the assay of adenosine triphosphate (ATP). For the Exo III-assisted target recycling amplification, DNA and ATP were fast detected with high sensitivity and selectivity. This work provides opportunities to develop simple, rapid, economical, and sensitive biosensing platforms for biomedical diagnostics.

The strong fluorescence quenching ability towards a wide spectrum of fluorescent dyes of bioinspired polydopamine nanospheres was shown for the first time. Up to 97% quenching efficiency via energy transfer and/or electron transfer was obtained towards four kinds of fluorophores, aminomethylcoumarin acetate (AMCA), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA) and Cy5. This fluorescence quenching ability compared favorably with that of graphene oxide, the superquencher. The nanospheres (NS) also exhibit different affinities for various ssDNA conformations. Furthermore, FAM-labeled ssDNA was adsorbed onto NS through non-covalent binding to form an ssDNA/NS complex, leading to the quenching of the fluorescence of FAM. This complex was used as a sensing platform for the detection of DNA and proteins based on the fluorescence recovery due to target recognition. The LODs of DNA and thrombin were equal or close to those of GO-based biosensors. The assay is fast, simple and homogeneous, and could be used for fluorescence imaging. The excellent biocompatibility and biodegradability of polydopamine also render it suitable for in vivo applications.

Nanomaterials as tracing tags have been widely used in biosensors with high sensitivity and selectivity. In this work, a signal amplification electrochemical aptamer sensing strategy for the detection of protein was designed by combining the hybridization-inducing aggregate of DNA-functionalized silver nanoparticles (AgNPs) and differential pulse stripping voltammetry (DPSV) detection. The multiprobes containing hybridization DNA and aptamers were anchored onto the silver nanoparticles. The protein assay was prepared through the immobilization of capture aptamer that specifically recognizes platelet-derived growth factor (PDGF-BB) on gold nanoparticles modified screen-printed electrode (SPE) array. After a sandwich-type reaction, two kinds of DNA-modified AgNPs were simultaneously added on the electrode surface for specifically recognizing PDGF-BB and forming the AgNPs aggregate caused by in situ hybridization of DNA. Compared to the signal-labeled tag, the tracing aggregate tags showed a strong electroactivity for signal amplification through stripping detection of silver after preoxidation. By using the hybridization-inducing aggregate as electrochemical readouts, the sensor showed wide linear range and low detection limit. The hybridization-inducing AgNPs aggregate were further used as tracing tags in multiplied proteins assays for PDGF-BB and thrombin by using the SPE array chip as sensing platform. The cross-talk between different aptamer-modified electrodes on the same array was avoided because of the advantage of labeled AgNPs. The array detection was also applied in the logic gate operation. The proposed method described here is ideal for multianalytes determination in clinical diagnostics with good analytical performance.

•A novel assay method based on FRET between fluorescent SiNPs and AuNPs was developed.•The method was based on the conformation change of aptamer lead by target recognition.•The aptamer toward adenosine without modification was split into two fragments.•The method was label-free without the modification of fluorescent SiNPs or AuNPs.•The detection limit of adenosine was as low as 45 nM with excellent specificity.A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.

•The silver decahedral nanoparticles (Ag10NPs) are very stable in NaCl.•The functionalization of Ag10NPs with ssDNA is rapid, just 2 h.•The prepared colorimetric sensor based on aptamer-Ag10NPs is highly sensitive and selective.•The sensor can be applied in detection of PDGF-BB in serum.Aptamer-silver decahedral nanoparticles (Ag10NPs-aptamer) based detection was developed for protein. Ag10NPs were synthesized by photochemical method. The advantage of Ag10NPs was its tolerance of NaCl which facilitates the functionalization of silver nanoparticles with all kinds of ssDNA. Attaching aptamers to Ag10NPs could be achieved within 2 h, much faster than traditional methods. Human platelet-derived growth factor-BB (PDGF-BB) was used as a model protein to test the binding capacity of aptamers attached on Ag10NPs. Our data showed that the aptamer-Ag10NPs conjugates were successful in detecting human PDGF-BB. Furthermore, we developed an aptamer-Ag10NPs conjugates-based colorimetric sensor to detect PDGF-BB. The results showed a linear relationship between PDGF-BB concentrations (5 ng mL−1–200 ng mL−1) and ΔOD with excellent detection specificity in serum. Therefore, the sensor based on aptamer-Ag10NPs conjugates was highly effective and sensitive and had great promise for further development and applications.

•A scanometric assay system based on apt-AgNPs and HAuCl4 was first developed.•The signal can be easily gotten by human eyes and flatbed scanner.•The apt-AgNPs system showed remarkable superiority compared to apt-AuNPs system.•The apt-AgNPs system had a wider linear range and good specificity in human serum.In this work, we reported a scanometric assay system based on the aptamer-functionalized silver nanoparticles (apt-AgNPs) for detection of platelet-derived growth factor-BB (PDGF-BB) protein. The aptamer and ssDNA were bound with silver nanoparticles by self-assembly of sulfhydryl group at 5′ end to form the apt-AgNPs probe. The apt-AgNPs probe can catalyze the reduction of metallic ions in color agent to generate metal deposition that can be captured both by human eyes and a flatbed scanner. Two different color agents, silver enhancer solution and color agent 1 (10 mM HAuCl4 + 2 mM hydroquinone) were used to develop silver and gold shell on the surface of AgNPs separately. The results demonstrated that the formation of Ag core–Au shell structure had some advantages especially in the low concentrations. The apt-AgNPs probe coupled with color agent 1 showed remarkable superiority in both sensitivity and detection limit compared to the apt-AuNPs system. The apt-AgNPs system also produced a wider linear range from 1.56 ng mL−1 to 100 ng mL−1 for PDGF-BB with the detection limit lower than 1.56 ng mL−1. The present strategy was applied to the determination of PDGF-BB in 10% serum, and the results showed that it had good specificity in complex biological media.

•An AgNP-based non-aggregation colorimetric aptasensor was first developed.•The colorimetric principle was based on AgNP-catalyzed reductive degradation of RhB.•This assay combined magnetic separation with nanocatalytic amplification.•The detection limit of thrombin was as low as 0.2 nM with excellent specificity.In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well.

This paper presents an ultrasensitive fluorescent detection method through fabricating a silver microarray substrate. Silver nanoparticles (AgNPs) and Ag@Au core–shell nanoparticles with different sizes were first synthesized by a seed-mediated growth method and the metal-enhanced fluorescence of these nanoparticles on different fluorescent dyes was investigated. The results indicated that AgNPs could act as a versatile and effective metal-enhanced fluorescence material for various fluorophores, whereas the enhanced fluorescence from Ag@Au was limited only to certain fluorophores. When the AgNPs were functionalized with aptamers and fluorescent dyes, a good analytical performance for simultaneous detection of human IgE and platelet-derived growth factor-BB (PDGF-BB) could be obtained. AgNPs were not only used as detection tags but also used to fabricate the plasmonic microarray substrate to further enhance the sensitivity of fluorescent detection. As a result, a linear response to PDGF-BB concentration was obtained in the concentration range of 16 pg mL−1 to 50 ng mL−1, and the detection limit was 3.2 pg mL−1. In addition, the AgNP modified plasmonic microarrays showed remarkable recovery and no significant interference from human serum when applied to 2 ng mL−1 PDGF-BB concentration. The plasmonic microarray substrate demonstrated both high specificity and sensitivity for protein microarray detection and this novel approach has great potential for ultrasensitive detection of protein biomarkers in the bio-medical field.

•A turn-off, AuNP-catalyzed amplified colorimetric thrombin aptasensor is presented.•Reductive bleaching of colored substrate is used as a novel indicator reaction.•We explore its fundamental principle and amplification factor theoretically.•Endpoint-based colorimetric assay was achieved using 4-NP as the substrate.•Kinetic-based colorimetric assay was first achieved using MB as the substrate.Nanoparticle-catalyzed reductive bleaching reactions of colored substrates are emerging as a class of novel indicator reactions for fabricating enzyme-free amplified colorimetric biosensing (turn-off mode), which are exactly opposite to the commonly used oxidative coloring processes of colorless substrates in traditional enzyme-catalyzed amplified colorimetric bioassays (turn-on mode). In this work, a simple theoretical analysis shows that the sensitivity of this colorimetric bioassay can be improved by increasing the amplification factor (kcatΔt), or enhancing the binding affinity between analyte and receptor (Kd), or selecting the colored substrates with high extinction coefficients (ε). Based on this novel strategy, we have developed a turn-off and cost-effective amplified colorimetric thrombin aptasensor. This aptasensor made full use of sandwich binding of two affinity aptamers for increased specificity, magnetic particles for easy separation and enrichment, and gold nanoparticle (AuNP)-catalyzed reductive bleaching reaction to generate the amplified colorimetric signal. With 4-nitrophenol (4-NP) as the non-dye colored substrate, colorimetric bioassay of thrombin was achieved by the endpoint method with a detection limit of 91 pM. In particular, when using methylene blue (MB) as the substrate, for the first time, a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of acidification termination and magnetic removal of particles, with a low detection limit of 10 pM, which was superior to the majority of the existing colorimetric thrombin aptasensors. The proposed colorimetric protocol is expected to hold great promise in field analysis and point-of-care applications.

•We briefly discuss the properties of silver nanoparticles (AgNPs).•We mention functionalization methods for AgNPs.•We describe applications of AgNPs in DNA, miRNA, protein and cell analysis.•Silver labels are highly sensitive in bioassays.•We discuss detection methods for silver labels.Silver nanoparticles (AgNPs) have attracted increasing attention for applications in bioanalytical chemistry because of their unique optical, electrical, chemical and catalytic properties. The review describes the properties of AgNPs and covers recent developments in bioanalytical applications based on the electrochemical, optical and catalytic properties of AgNPs.

A silver nanoparticle (AgNP)-enhanced fluorescence resonance energy transfer (FRET) sensing system is designed for the sensitive detection of human platelet-derived growth factor-BB (PDGF-BB). Fluorophore-functionalized aptamers and quencher-carrying strands hybridized in duplex are coupled with streptavidin (SA)-functionalized nanoparticles to form a AgNP-enhanced FRET sensor. The resulting sensor shows lower background fluorescence intensity in the duplex state due to the FRET effect between fluorophores and quenchers. Upon the addition of PDGF-BB, the quencher-carrying strands (BHQ-2) of the duplex are displaced leading to the disruption of the FRET effect. As a result, the fluorescent intensity of the fluorophore–aptamer within the proximity of the AgNP is increased. When compared to the gold nanoparticle (AuNP)-based FRET and bare FRET sensors, the AgNP-based FRET sensor showed remarkable increase in fluorescence intensity, target specificity, and sensitivity. Results also show versatility of the AgNP in the enhancement of sensitivity and selectivity of the FRET sensor. In addition, a good linear response was obtained when the PDGF-BB concentrations are in the ranges of 100–500 and 6.2–50 ng/mL with the detection limit of 0.8 ng/mL.

In this report, Ag nanoparticles/graphene hybrid (AgNPs/graphene) was fabricated according to a self-assembly procedure followed by functionalization with streptavidin (SA) and was used as electrochemical label in the electrochemical detection of human immunoglobulin E (IgE) through a sandwich-type strategy. The thiol-capped IgE DNA aptamer was used as capture probe, while the SA functionalized AgNPs/graphene (SA-AgNPs/graphene) linked with biotinylated goat anti-human IgE antibody was used as detection probe. The electrochemical signal from AgNPs anchored on the graphene was obtained through square wave anodic stripping voltammetry. The results demonstrated that this electrochemical biosensor possessed a dynamic range from 10 to 1000 ng mL− 1 with a low detection limit of 3.6 ng mL− 1.Highlights► Silver nanoparticles/graphene hybrid (AgNPs/graphene) was fabricated. ► The AgNPs/graphene was functionalized with streptavidin. ► The AgNPs/graphene was used as electrochemical label for the determination of IgE.

An ultrasensitive, fast and specific fluorescent platform for protein detection is developed. In this protocol, silver nanoparticles were conjugated with paramagnetic particles (MPs–Ag) for target capture, concentration and separation; fluorescent dyes functionalized silver nanoparticles (Tag) for generating signals. The presented method is highly sensitive and specific with a detection limit of 2.2 pM for thrombin, and no significant interference was observed for other proteins such as human serum albumin (HSA), lysozyme and IgG. This novel approach combining the magnetic separation and concentration of MPs–Ag, aptamer recognition and fluorescence enhancement of Tag, can be successfully used to enhance the sensitivity of detecting ultra-low levels of target proteins or biomolecules.

A novel, enzyme-free and aptamer-based colorimetric platform for protein detection has been developed, which takes advantage of aptamer-functionalized magnetic beads (MBs) for target capture, concentration and separation, and aptamer-conjugated gold nanoparticle (AuNP)-catalyzed color bleaching reaction of methyl orange (MO) to generate the colorimetric signals. It was demonstrated that the proposed colorimetric sensing strategy enables simple, cost-effective, sensitive and specific thrombin detection without the use of any enhancing solutions and enzymes. Herein, by naked eye observation, we can detect the human thrombin with a detection limit of approximately 320 pM, which can be further decreased to 30 pM with the help of a UV-vis instrument. In addition, this method also works for targets with two or more binding sites.

A novel biosensor array for DNA detection based on the coupling of silver nanoparticles is developed. Two kinds of DNA–silver nanoparticle conjugates were immobilized on the aldehyde modified slides. The fluorescence intensity of Cy3 within the proximity of the conjugates was enhanced due to the coupling of the silver nanoparticles based on hybridization of target DNA and the conjugates. The fluorescence intensity increases with the concentration of target DNA, and the enhancement factor is about 2.4 at the optimized conditions. The results gave a good linear correlation between DNA concentration and fluorescence intensity at DNA concentration range from 12.8 pM to 40 nM. The biosensors constructed on the slide as a microarray offer a unique approach for DNA detection with the advantages of high sensitivity and rapidity. The fluorescence enhancement was attributed to the coupling between the silver nanoparticles. This new strategy opens the possibility for the preparation of highly enhanced plasmonic substrate for DNA analysis.

In the present work we design a novel aptamer-based silver nanosensor for one-spot simultaneously detection of multiple proteins. SS-DNA modified AgNPs were immobilized on the aldehyde coated glass slide to form an AgNP array. Then dye-labeled aptamer sequences were allowed to hybridize with their complementary strands assembled on the surface of AgNPs. The target proteins were introduced to associate with the corresponding aptamers to form the aptamer–target complexes. The removal of the aptamer–target complexes resulted in a remarkable decrease in fluorescent signals. This nanosensor is found to be highly sensitive for the detection of proteins. When thrombin was employed as a sample model, the limit of detection of the optimized nanosenor was 0.4 fmol with a linear response of 0.8 fmol to 0.5 pmol. We further demonstrated the multiple protein detection of IgE and thrombin using multicolor silver nanoprobes, which shows effective recognition of the relative protein individually or simultaneously. This silver nanosensor offers a unique heterogeneous approach for protein detection with several advantages, such as high sensitivity, rapidity, high throughput, and miniaturization.

We present a highly sensitive metal enhanced fluorescence (MEF) method based on a novel silver nanostructure fabricated with Cy5-functionalized silver nanoparticles (AgNPs) and AgNO3. The analytical performance has been demonstrated by microarray detection of streptavidin (SA) and human IgE. The fluorescence intensity can be enhanced substantially with the combined use of AgNPs and fluorescence enhanced solution (FES). Aptamers have been used for the preparation of Tag-C, which demonstrate IgE detection from 0.5 ng/mL to 16 ng/mL, and the limit of detection is determined to be 0.25 ng/mL. SEM images show nanogaps exist in the aggregated silver nanoparticles and the nanogaps allow for the trap of fluorophores in the nanostructures that emit brighter light upon excitation. The silver nanostructures formed by Tags and FES proved to be an excellent platform for MEF of fluorophores whose excitation and emission occurred between 436 nm and 1000 nm. Finite-difference time-domain (FDTD) simulation has been carried out to confirm the enhanced electromagnetic field inside silver nanostructures, leading to strong overlap/resonance coupling and eventual fluorescence enhancement.

In the present study, we report a novel aptasensor based on silver nanoparticle enhanced fluorescence for the detection of adenosine. First, the distance dependence nature of silver nanoparticle enhanced fluorescence was investigated through fluorescent dyes modified oligonucleotides to control the spacing distance between dyes and AgNP. The results showed that the fluorescence intensity reached the maximum value with the spacing distance of dyes about 8 nm from AgNP surface. The fluorescence intensity decreases when the spacing distance is either above or below this value. Based on this result, a fluorescence switch is constructed. In the “OFF” state, without the target molecules, there is a greater spacing distance between the Cy3 dyes and the AgNP giving comparatively lower fluorescence intensity. While in the “ON” state, in the presence of target molecules, the fluorescence signals increased for the conformation structure change of the aptamer which shorten the spacing distance between the Cy3 dyes and the AgNP to 8 nm. Using adenosine as target, the aptasensor produced a linear range from 200 nM to 200 μM with a correlation coefficient of 0.9949 and the detection limit was 48 nM estimated using 3σ. The aptasensor was also found to be specific in targeting adenosine. The presented method shows a new strategy of combining aptamer recognition and silver nanoparticle for fluorescence signal enhancement and increasing sensitivity.Highlights► Aptasensor designed based on MEF effect of AgNPs. ► Distance dependence nature of MEF investigated by Cy3 modified oligonucleotides. ► Conformation change of aptamers changes the fluorescence of Cy3 on the AgNPs. ► A fluorescence switch constructed for detection of adenosine.

The estrogen receptor (ER) is regarded as a significant drug target because of its important physical and pathological function. In this article, we describe a novel screening method to obtain agonists and antagonists of ER. ER was immobilized onto an aldehyde-modified glass slide. The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined. Two libraries, one containing 29 synthetic compounds and the other with 384 natural products that served as a model, were screened to find new ligands for ER. The IC50 values obtained for tamoxifen and raloxifene were consistent with those found in the literature (4.85 × 10−7 M versus 1.74~4.23 × 10−7 M and 7.58 × 10−8 M versus 0.89~5.84 × 10−8 M, respectively). Finally, 65 active ligands (5 synthetic compounds and 60 natural products) of ER were identified. This novel method gave identical results to a conventional fluorescence polarization assay, thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays. The presented method is sensitive, accurate, and reliable, and shows great potential for use in high-throughput drug-screening research.

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.

An ultrasensitive protein assay method was developed based on silver nanoparticle (AgNP) hybrid probes and metal-enhanced fluorescence. Two aptamer based silver nanoparticles, Aptamer/Oligomer-A/Cy3-modified AgNPs (Tag-A) and Aptamer/Oligomer-B/Cy3-modified AgNPs (Tag-B) were hybridized to form a silver nanoparticle aggregate that produced a red shift and broadening of the Localized Surface Plasmon Resonance (LSPR) peak. The enhanced fluorescence resulted from the increased content of Cy3 molecules and their emission resonance coupled to the broadened localized surface plasmon (LSP) of AgNP aggregate. The separation distance between Cy3 and AgNPs was 8 nm which was the most optimal for metal enhanced fluorescence and the separation distance between adjacent AgNPs was about 16 nm and this was controlled by the lengths of oligomer-A and oligomer-B. The protein array was prepared by covalently immobilizing capture antibodies on aldehyde-coated slide. After addition of protein IgE sample, two kinds of aptamer-modified AgNPs (Tag-A and Tag-B) were employed to specifically recognize IgE and form the AgNP aggregate on the arrays based on their hybridization. The detection property of the aptamer-modified AgNP aggregate was compared to two other modified aptamer-based probes, aptamer-modified Cy3 and Tag-A. The modified AgNP hybrid probe (Tag-A and Tag-B) showed remarkable superiority in both sensitivity and detection limit due to the formed AgNP aggregate. The new hybrid probe also produced a wider linear range from 0.49 to 1000 ng/mL with the detection limit reduced to 40 pg/mL (211 fM). The presented method showed that the newly designed strategy of combining aptamer-based nanomaterials to form aggregates results in a highly sensitive optical detection method based on localized surface plasmon.

In this work a novel microdevice sensor has been developed by plating gold on the PDMS surface to generate a sandwich-type gap electrode for DNA detection. The microdevice utilizes a gold band electrode–PDMS–gold band electrode configuration and the minimum detectable volume could be as low as 5 μL. The 20 μm PDMS-based gap was chemically modified with DNA capture probes and DNA sandwich hybrids were formed with the addition of DNA target and silver nanoparticle probes. To increase detection sensitivity, parallel detection zones have been developed in which the relevant resistances decrease substantially upon hybridyzation. By measuring the change in electrical conductivity, the DNA target in the concentration range of 1000–0.1 nM can be assayed and the limit of lowest detectable concentration was achieved at 0.01 nM.

In this article, an electrochemically direct stripping approach based on silver nanoparticles (AgNPs) labeled with antibody was proposed. To prepare AgNPs labels, glutathione (GSH) was chemically absorbed on the surface of AgNPs through its free thiol groups. Glutaraldehyde was used as a coupling reagent, its two aldehyde groups reacted with the amino group of GSH absorbed on the AgNPs surfaces and the amino groups of antibodies, respectively. The resulting labeled electrochemical active nanoparticles could recognize the proteins specifically via the immobilized antibody. To prove the electrochemical property of labeled AgNPs, human IgG was used as a model protein sample to be immobilized on a screen printed electrode (SPE) and the goat-anti-human IgG labeled AgNPs was added onto the surface of the SPE, followed by differential pulse voltammetric (DPV) method. With the oxidation of AgNPs labels coupled on the electrodes, the concentration of hIgG could be assayed directly and calculated. The dynamic concentration was in the range of 1–1000 ng/mL and the detection limit was 0.4 ng/mL (S/N = 3). In addition, the presented method was also compared to indirect electrochemical stripping detection by dissolving AgNPs labels with nitric acids and the results showed advantages such as lower detection limit, rapidity and simplicity.Research highlights► In this article an electrochemically direct stripping approach based on silver nanoparticles (AgNPs) labeled with antibody was proposed. ► Human IgG was used as a model protein sample to be immobilized on a screen printed electrode and the goat-anti-human IgG labeled AgNPs was added onto the surface of the SPE. ► With the oxidation of AgNPs labels, the concentration of hIgG could be assayed directly and calculated. ► The dynamic concentration was in the range of 1–1000 ng/mL and the detection limit was 0.4 ng/mL (S/N = 3).

Multiplexed DNA target detection is of great significance in many fields including clinical diagnostics, environmental monitoring, biothreat detection and forensics. Although the emergence of DNA chip technology has accelerated this process, it is still a challenge to perform ultrasensitive DNA assay at low attomol concentrations so that DNA detection can be directly achieved without a PCR protocol. In this work, an oligonucleotide-functionalized silver nanoparticle tag has been successfully developed for multiplexed DNA electrochemical detection with ultrahigh sensitivity. The multiprobes containing oligo(d)A and the reporting probes were anchored onto the silver nanoparticles, followed by hybridizing with the silver nanoparticle conjugate modified with oligo(d)T. The hybridization-induced tag was found to show an aggregated nanostructure 10 times larger than the individual nanoparticle, as revealed by TEM. For sandwich-based assays, the tag was specifically coupled to a gold electrode surface via target DNA. Compared to a single nanoparticle label, this novel tag has shown excellent electroactive property and produces 103-fold amplification in the differential pulse voltammetric (DPV) method. Hepatitis B virus (HBV) sequence was employed as a sample model, and we have achieved a detection limit of 5 aM (∼120 molecules in 40 μL volume), demonstrating ultrasensitive measurement for DNA. The property of the electrochemical process involving silver aggregates was further investigated and the integrative oxidation of the silver tag was observed. We further demonstrated the multiplexed DNA target detection using array chips functionalized with Herpes simplex virus (HSV), Epstein−Barr virus (EBV) and cytomegalovirus (CMV) sequences, which shows effective recognition of the relative sequences individually or simultaneously. The method offers a uniquely new approach for DNA detection with ultrahigh sensitivity as well as advantages of rapidity, throughput, and miniaturization.

A core-shell Rhodamine B-doped SiO2 nanoparticle was synthesized and its fluorescent intensity was found to be 1000 times higher than that of individual Rhodamine B molecule. The doped nanoparticles were further conjugated with streptavidin and the resulting nanoparticles were used in the detection of reverse-phase protein microarrays, in which human IgG of various concentrations was first immobilized on aldehyde-modified glass slides and then biotinlyated goat anti human IgG as well as the labeled nanoparticles were sequentially conjugated. The calibration curve is linear over the range from 800 fg to 500 pg and the limit of detection is 100 fg, which is 8 times lower than that of streptavidin-labeled Cy3 fluorescent dyes. The dye-doped SiO2 nanoparticles show potentials for the protein array detection.

A novel protein assay method based on a DNA array was developed, in which human immunoglobulin E (hIgE) and its DNA aptamer were used as an analytical model. The target protein hIgE was captured by the aptamer in homogeneous solution and then the resulting hIgE-aptamer complex was hybridized onto probes self-assembled on the DNA array. Measured by electrochemical impedance spectroscopy (EIS), the charge transfer resistance (Rct) of electrodes before and after hybridization was compared. To test the selectivity of the method, four different probes with one to three mismatched bases were immobilized on respective electrodes. The results showed that the complex could be hybridized and detected out on the electrodes modified with the fully complementary sequences. In addition, the DNA array could be employed to analyze multiple samples selectively with the matched aptamer.

A novel biosensor array for DNA detection based on the coupling of silver nanoparticles is developed. Two kinds of DNA–silver nanoparticle conjugates were immobilized on the aldehyde modified slides. The fluorescence intensity of Cy3 within the proximity of the conjugates was enhanced due to the coupling of the silver nanoparticles based on hybridization of target DNA and the conjugates. The fluorescence intensity increases with the concentration of target DNA, and the enhancement factor is about 2.4 at the optimized conditions. The results gave a good linear correlation between DNA concentration and fluorescence intensity at DNA concentration range from 12.8 pM to 40 nM. The biosensors constructed on the slide as a microarray offer a unique approach for DNA detection with the advantages of high sensitivity and rapidity. The fluorescence enhancement was attributed to the coupling between the silver nanoparticles. This new strategy opens the possibility for the preparation of highly enhanced plasmonic substrate for DNA analysis.

The strong fluorescence quenching ability towards a wide spectrum of fluorescent dyes of bioinspired polydopamine nanospheres was shown for the first time. Up to 97% quenching efficiency via energy transfer and/or electron transfer was obtained towards four kinds of fluorophores, aminomethylcoumarin acetate (AMCA), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA) and Cy5. This fluorescence quenching ability compared favorably with that of graphene oxide, the superquencher. The nanospheres (NS) also exhibit different affinities for various ssDNA conformations. Furthermore, FAM-labeled ssDNA was adsorbed onto NS through non-covalent binding to form an ssDNA/NS complex, leading to the quenching of the fluorescence of FAM. This complex was used as a sensing platform for the detection of DNA and proteins based on the fluorescence recovery due to target recognition. The LODs of DNA and thrombin were equal or close to those of GO-based biosensors. The assay is fast, simple and homogeneous, and could be used for fluorescence imaging. The excellent biocompatibility and biodegradability of polydopamine also render it suitable for in vivo applications.