Target-triggered tertiary amplifications for sensitive and label-free protein detection based on lighting-up RNA aptamer transcriptions

Microscale thermophoresis sensor for homogeneous and one-step protein detection based on dual aptamers binding induced DNA switch.

Label-free and homogeneous aptamer proximity binding assay for fluorescent detection of protein biomarkers in human serum

We introduce here a novel assay for the detection of platelet-derived growth factor BB (PDGF-BB) via hybridization chain reaction (HCR) based on an aptameric system, where stable DNA monomers assemble only upon exposure to a target PDGF-BB aptamer. In this process, two complementary stable species of biotinylated DNA hairpins coexist in solution until the introduction of initiator aptamer strands triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers. In detail, the aptamer firstly opens the hairpins in the solution, creating long concatemers, and then reacts with the antibody captured PDGF-BB on the well surface. Moreover, several experimental conditions including different PDGF-BB aptamers, the spacer length of the selected aptamer and hairpin, etc. are investigated and optimized. Our results show that the coupling of HCR to aptamer triggers for the amplification detection of PDGF-BB achieves a better performance in the fluorescence detection of PDGF-BB as compared to the traditional antibody-antigen-aptamer assays. Upon modification, the approach presented herein could be extended to detect other types of targets. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment and environmental monitoring.

A biolayer interferometry-based enzyme-linked aptamer sorbent assay for real-time and highly sensitive detection of PDGF-BB

Herein, a general protein conversion and analysis strategy was developed for homogeneous, label-free, and sensitive protein detection, on the basis of the affinity binding-induced Hg2+ release for protein conversion, and the succeeding Hg2+ doping-induced ZnSe quantum dot (QD) photoluminescence for signal readout. Two DNA motifs were designed, each of which was conjugated with a protein-specific recognition ligand. The mercury ions were initially introduced into one DNA motif by T-Hg2+-T interaction. The Hg2+ releasing was then accomplished after protein recognition-initiated strand exchange reaction between two DNA motifs. Then, the simultaneous incorporation of the released Hg2+ into ZnSe QD resulted in a doping-dependent fluorescence emission at 560 nm correlated with protein analysis. The protein assay is outperformed only by a simple one-step mixing operation but no separation or washing steps. Also, the use of doped QD as a fluorogenic reporter can avoid the fluorophore and/or quencher labeling, and eliminate complex DNA manipulation procedures for signal readout or amplification involved in most existing nucleic acid-based protein conversion and analysis methods. The versatile and sensitive detection toward multivalent proteins was verified with the detection limits achieved at 0.034 nM for anti-Dig antibody, 0.012 nM for streptavidin, and 0.025 nM for thrombin. Thus, it shows great promise for protein analysis to accommodate the applications in disease diagnosis, biomarker screening, and clinical medicine.

An innovative surface-enhanced Raman spectroscopy and lateral flow assay (SERS-LFA) biosensor combined with aptamer recognition had been developed for the convenient, rapid, sensitive and accurate detection of thrombin and platelet-derived growth factor-BB (PDGF-BB) associated with prostate cancer simultaneously. During the biosensor operation, thrombin and PDGF-BB in the sample were recognized and combined by thiol-modified aptamers immobilized on Au–Ag hollow nanoparticles (Au–Ag HNPs) surface and biotinylated aptamers immobilized on the test lines of the biosensor. Thus, thrombin and PDGF-BB were simultaneously captured between detection aptamers and capture aptamers in a sandwich structure. Finite difference time domain simulation confirmed that ‘hot spots’ appeared at the gaps of Au–Ag HNPs dimer in the enhanced electromagnetic field compared to that of a single Au–Ag HNP, indicating that the aggregated Au–Ag HNPs owned a good SERS signal amplification effect. The detection limits of thrombin and PDGF-BB in human plasma were as low as 4.837 pg ml−1 and 3.802 pg ml−1, respectively. Moreover, the accuracy of the biosensor which was applied to detect thrombin and PDGF-BB in prostate cancer plasma had been verified. This designed biosensor had broad application prospects in the clinical diagnosis of prostate cancer.

Here we report a lateral flow aptasensor (LFA) for the simultaneous detection of platelet-derived growth factor-BB (PDGF-BB) and thrombin. Two pairs of aptamers, which are specific against PDGF-BB and thrombin, respectively, were used to prepare the LFA. Thiolated aptamers were immobilized on a gold nanoparticle (AuNP) surface and biotinylated aptamers were immobilized on the test zones of an LFA nitrocellulose membrane. The assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers on the test zones of LFA and AuNP-labeled detection aptamers. AuNPs were thus captured on the test zones of the LFA and gave red bands to enable the visual detection of target proteins. Quantitative results were obtained by reading the test band intensities with a portable strip reader. By combining the highly specific molecular recognition properties of aptamers with the unique properties of lateral flow assay (low-cost, short assay time and a user-friendly format), the optimized aptasensor was capable of simultaneously detecting 1.0 nM of PDGF-BB and 1.5 nM of thrombin in association with a 10-min assay time. The biosensor was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers.

Alpha-fetoprotein (AFP) is a tumor-associated fetal protein that can be expressed in large amounts in adult tumor cells, serving as a useful clinical tumor-marker. Silicon nanowire (SiNW) biosensors have emerged as a powerful tool in detecting protein biomarkers, due to their ultrahigh sensitivity, real-time response and label-free detection. We fabricated a SiNW-based field-effect transistor (FET) according to “top-down” methodology. First, anti-AFP antibodies were immobilized onto the surface of the SiNW-FET. A polydimethylsiloxane (PDMS) microchannel was then integrated to the modified SiNW-FET. Various concentrations of AFP were then pumped through the sensing area. We observed a current change that corresponded to binding of AFP onto the surface of our anti-AFP functionalized SiNW-FET biosensor. Concentrations of AFP as low as 0.1 ng/mL were detected. The results implicate our SiNW biosensor as an effective AFP biomarker detector with promising potential in clinical applications.

A sensitive and recyclable fluorescence aptasensor for detection and extraction of platelet-derived growth factor BB

Portable aptamer biosensor of platelet-derived growth factor-BB using a personal glucose meter with triply amplified

Electrochemiluminescent aptasensor based on β-cyclodextrin/graphitic carbon nitride composite for highly selective and ultrasensitive assay of platelet derived growth factor BB

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [Ru(phen)2(dppz)]2+. The method takes advantage of the sensitive luminescence signal change of [Ru(phen)2(dppz)]2+ intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [Ru(phen)2(dppz)]2+/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and alpha-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.

Signal transducer and activator of transcription 3—A key molecular switch for human mesenchymal stem cell proliferation

The authors describe a dual signal amplification strategy for improving the sensitivity of electrochemical aptasensor. Hydroxyapatite nanoparticles (HAP-NPs) serve as the support for deposition of the respective aptamer. Both the HAP-NPs and the aptamer contain phosphate groups which can react with molybdate to form a redox-active molybdophosphate precipitate on the surface of a glassy carbon electrode (GCE). On applying a relatively low voltage of 0.21 V (vs. Ag/AgCl), a current is generated whose intensity depends on the concentration of the analyte. The cancer biomarker platelet-derived growth factor BB (PDGF-BB) is chosen as a model antigen (analyte). The assay works by sequential deposition of antibody against PDGF-BB, analyte (PDGF-BB) and anti-PDGF-BB aptamer modified HAP-NPs on the GCE to form a sandwich structure. The amperometric signal is linear in the 0.1 pg.mL−1 to 10 ng.mL−1 PDGF-BB concentration range, with a detection limit as low as 50 fg.mL−1. The assay was successfully applied to the determination of PDGF-BB in serum samples. In our perception, this signal amplification strategy has a wide scope in that it can be adapted to the preparation of other aptasensors for biomarkers and related species.

BACKGROUND: Globally, severe acute respiratory syndrome coronavirus (SARS‐CoV) is a newly emerging virus that causes SARS with high mortality rate in infected people. The nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)‐associated coronavirus (SARS‐CoV) is an important antigen for the early diagnosis of SARS and the detection of diseases. Here, a new quantum dots (QDs)‐conjugated RNA aptamer with high sensitivity and rapidity is proposed for the detection of SARS‐CoV N protein using an on chip system. RESULTS: A QDs‐conjugated RNA aptamer can specifically hybridize on the immobilized SARS‐CoV N protein on the surface of a glass chip. Detection is based on the optical signal variation of a QDs‐supported RNA aptamer interacting on an immobilized protein chip. Using an optical QDs‐based RNA aptamer chip, SARS N protein was detected at concentrations as low as 0.1 pg mL−1. CONCLUSIONS: It was demonstrated that the QDs‐conjugated RNA aptamer could interact on a designed chip specifically and sensitively. This device could form a QDs‐conjugated biosensor prototype chip for SARS‐CoV N protein diagnosis. The proposed visual SARS‐CoV N protein detection technique may avoid the limitations of other reported methods because of its high sensitivity, good specificity, ease of use, and the ability to perform one‐spot monitoring. Copyright © 2011 Society of Chemical Industry