Herein we demonstrate a sensitive approach for protein detection based on peak shifts of localized surface plasmon resonance (LSPR) induced by aptamer-antigen-antibody sandwich structures. The applicability of the proposed method is demonstrated using human α-thrombin as a model analyte. While the binding of thrombin to its specific receptor, thrombin binding aptamer (TBA) modified on Au nanorods (AuNRs), causes a measurable LSPR shift, a subsequent binding of an anti-thrombin antibody to the captured thrombin can exhibit a nearly 150% amplification in the LSPR response. This enhanced signal essentially leads to an improvement of limit of detection (LOD) by more than one order of magnitude. In addition, the use of TBA as thrombin recognition units makes the biosensor reusable. The feasibility of the proposed method was further exploited by the detection of thrombin in human serum, opening the possibility of a real application for diagnostics and medical investigations.
[Show abstract][Hide abstract] ABSTRACT: Nanoimprinting lithography (NIL) is a manufacturing process that can produce macroscale surface areas with nanoscale features. In this paper, this technique is used to solve three fundamental issues for the application of localized surface plasmonic resonance (LSPR) in practical clinical measurements: assay sensitivity, chip-to-chip variance, and the ability to perform assays in human serum. Using NIL, arrays of 140 nm square features were fabricated on a sensing area of 1.5 mm x 1.5 mm with low cost. The high reproducibility of NIL allowed for the use of a one-chip, one-measurement approach with 12 individually manufactured surfaces with minimal chip-to-chip variations. To better approximate a real world setting, all chips were modified with a biocompatible, multi-component monolayer and inter-chip variability was assessed by measuring a bioanalyte standard (2.5-75 ng/ml) in the presence of a complex biofluid, human serum. In this setting, nanoimprinted LSPR chips were able to provide sufficient characteristics for a 'low-tech' approach to laboratory-based bioanalyte measurement, including: 1) sufficient size to interface with a common laboratory light source and detector without the need for a microscope, 2) high sensitivity in serum with a cardiac troponin limit of detection of 0.55 ng/ml, and 3) very low variability in chip manufacturing to produce a figure of merit (FOM) of 10.5. These findings drive LSPR closer to technical comparability with ELISA-based assays while preserving the unique particularities of a LSPR based sensor, suitability for multiplexing and miniaturization, and point-of-care detections.
PLoS ONE 03/2015; 10(3):e0120974. DOI:10.1371/journal.pone.0120974 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, a new type of rapid, high sensitive and selective fluorescence turn-on assay for detection of glutathione using an Alizarin Red S/copper ion ensemble is developed. This assay is based on the highly specific interaction between the glutathione and the copper ions and the strong fluorescence Alizarin Red S probe in a competition assay format. The system is simple in design, fast in operation and is more convenient and promising than other methods. The novel strategy eliminated the separation process, chemical modifications, and sophisticated instrumentations. The detection and discrimination process can be seen with the naked eye and can be easily adapted to automated high-throughput screening. The assay has high sensitivity and selectivity for glutathione. The detection limit is 2.3 nM, it is lower than or at least comparable to previous methods. The dynamic range of the sensor can be tuned simply by adjusting the concentration of copper ions. Importantly, the protocol offers high selectivity for the determination of glutathione among amino acids found in proteins, as well as in serum samples. The assay shows great potential for practical application as a disease-associated biomarker and it will be needed to satisfy the great demand of amino acid determination in the fields such as biochemistry, pharmaceuticals, and clinical analysis.
[Show abstract][Hide abstract] ABSTRACT: The impact of chiral compounds on pharmacological and biological processes is well known. With the increasing need for enantiomerically pure compounds, effective strategies for enantioseparation and chiral discrimination are in great demand. Herein we report a simple but efficient approach for the enantioselective determination of chiral compounds based on a localized surface plasmon resonance (LSPR) biosensor integrated with a microfluidic chip. A glass microfluidic chip with an effective volume of ∼0.75 μL was fabricated for this application. Gold nanorods (AuNRs) with an aspect ratio of ∼2.6 were self-assembled onto the surface of the inner wall of the chip to serve as LSPR transducers, which would translate the analyte binding events into quantitative concentration information. Human α-thrombin was immobilized onto the AuNR surface for enantioselective sensing of the enantiomers of melagatran. The proposed sensor was found to be highly selective for RS-melagatran, while the binding of its enantiomer, SR-melagatran, to the sensor was inactive. Under optimal conditions, the limit of detection of this sensor for RS-melagatran was found to be 0.9 nM, whereas the presence of 10 000-fold amounts of SR-melagatran did not interfere with the detection. To the best of our knowledge, this is the first demonstration of an LSPR-based enantioselective biosensor.
Lab on a Chip 07/2012; 12(20):3901-6. DOI:10.1039/c2lc40388a · 6.12 Impact Factor
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