Novel Aptasensor Platform Based on Ratiometric Surface-Enhanced Raman Spectroscopy

A novel aptasensor platform has been developed for quantitative detection of adenosine triphosphate (ATP) based on a ratiometric surface-enhanced Raman scattering (SERS) strategy. The thiolated 3′-Rox-labeled complementary DNA (cDNA) is first immobilized on the gold nanoparticle (AuNP) surface and then hybridizes with the 3′-Cy5-labeled ATP-binding aptamer probe (Cy5-aptamer) to form a rigid double-stranded DNA (dsDNA), in which the Cy5 and Rox Raman labels are used to produce the ratiometric Raman signals. In the presence of ATP, the Cy5-aptamer is triggered the switching of aptamer to form the aptamer–ATP complex, leading to the dissociation of dsDNA, and the cDNA is then formed a hairpin structure. As a result, the Rox labels are close to the AuNP surface while the Cy5 labels are away from. Therefore, the intensity of SERS signal from Rox labels increases while that from Cy5 labels decreases. The results show that the ratio between the Raman intensities of Rox labels and Cy5 labels is well linear with the ATP concentrations in the range from 0.1 to 100 nM, and the limit of detection reaches 20 pM, which is much lower than that of other methods for ATP detection and is also lower than that of SERS aptasensor for ATP detection. The proposed strategy provides a new reliable platform for the construction of SERS biosensing methods and has great potential to be a general method for other aptamer systems

Label-Free Photonic Crystal-Based β‑Lactamase Biosensor for β‑Lactam Antibiotic and β‑Lactamase Inhibitor

A simple, label-free, and visual photonic crystal-based β-lactamase biosensor was developed for β-lactam antibiotic and β-lactamase inhibitor in which the penicillinase (a β-lactamase) was immobilized on the pH-sensitive colloidal crystal hydrogel (CCH) film to form penicillinase colloidal crystal hydrogel (PCCH) biosensing film. The hydrolysis of penicillin G (a β-lactam antibiotic) can be catalyzed by penicillinase to produce penicilloic acid, leading to a pH decrease in the microenvironment of PCCH film, which causes the shrink of pH-sensitive CCH film and triggers a blue-shift of the diffraction wavelength. Upon the addition of β-lactamase inhibitor, the hydrolysis reaction is suppressed and no clear blue-shift is observed. The concentrations of β-lactam antibiotic and β-lactamase inhibitor can be sensitively evaluated by measuring the diffraction shifts. The minimum detectable concentrations for penicillin G and clavulanate potassium (a β-lactamase inhibitor) can reach 1 and 0.1 μM, respectively. Furthermore, the proposed method is highly reversible and selective, and it allows determination of penicillin G in fish pond water samples

Acetylcholinesterase Liquid Crystal Biosensor Based on Modulated Growth of Gold Nanoparticles for Amplified Detection of Acetylcholine and Inhibitor

A novel acetylcholinesterase (AChE) liquid crystal (LC) biosensor based on enzymatic growth of gold nanoparticles (Au NPs) has been developed for amplified detection of acetylcholine (ACh) and AChE inhibitor. In this method, AChE mediates the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, and the latter further reduces AuCl₄^– to Au NPs without Au nanoseeds. This process, termed biometallization, leads to a great enhancement in the optical signal of the LC biosensor due to the large size of Au NPs, which can greatly disrupt the orientational arrangement of LCs. On the other hand, the hydrolysis of ATCl is inhibited in the presence of ACh or organophosphate pesticides (OPs, a AChE inhibitor), which will decrease the catalytic growth of Au NPs and, as a result, reduce the orientational response of LCs. On the basis of such an inhibition mechanism, the AChE LC biosensor can be used as an effective way to realize the detection of ACh and AChE inhibitors. The results showed that the AChE LC biosensor was highly sensitive to ACh with a detection limit of 15 μmol/L and OPs with a detection limit of 0.3 nmol/L. This study provides a simple and sensitive AChE LC biosensing approach and offers effective signal enhanced strategies for the development of enzyme LC biosensors

Calibration curves for the relationship between the frequency shift of immunoreaction and the dilution ratio of <i>Sj</i>Ab.

<p>The dynamic dilution range of <i>Sj</i>Ab is ∼1∶1500 to 1∶60 with the detection limit of ∼1∶1800 dilution, estimated according to the 3σ (standard deviation) rule. A sample with 1∶100 dilution of <i>Sj</i>Ab was determined repeatedly for five times. The average response frequency shift value was 340±19 Hz, and the relative standard deviation (RSD) among five runs was 9.1%.</p

Effect of ME concentration in mixed SAM on <i>Sj</i>Ag immobilization.

<p>The best dose of ME in the mixed membrane was determined to be 70%.</p

Typical real time frequency response characteristics of the immunosensor in PBS solution.

<p>(a) NRS as negative control with PEG; (b) <i>Sj</i>Ab without PEG (dilution ratio 1∶100); (c) <i>Sj</i>Ab with PEG (dilution ratio 1∶100). Using PEG can significantly enhance the sensor response signals. Immune response-mediated frequency shift values increased from 225 Hz (b) to 343 Hz (c). The corresponding time of immune response decreased from 1600 s (b) to 1200 s (c). Considering the analysis time and reproducibility, the time for both analysis and detection of the sensors in this experiment was 1200 s.</p

Linear regression analysis of immunosensors and ELISA methods.

<p>The correlation coefficient is 0.973.</p

Effect of <i>Sj</i>Ag concentration on <i>Sj</i>Ag immobilization.

<p>The MPA and ME of volume ratios in mixed SAM are 3∶7. The <i>Sj</i>Ag dilution ratio is 1∶100. The best concentration of <i>Sj</i>Ag for immobilization is 0.2 mg ml⁻¹.</p

Modulated Dye Retention for the Signal-On Fluorometric Determination of Acetylcholinesterase Inhibitor

A novel fluorometric assay method based on target-induced signal on was developed for acetylcholinesterase (AChE) inhibitor with obviously improved detection sensitivity. In this method, the AChE molecules catalyzed the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, which in turn can specifically react with fluorescent squaraine derivative, a specific chemodosimeter for thiol-containing compounds, resulting in fluorescence quenching and offering a low fluorometric background for the further detection of AChE inhibitor. In the presence of AChE inhibitor, the catalytic hydrolysis of ATCl is blocked, and then the squaraine derivative remains intact and shows signal-on fluorescence. The amount of the remaining fluorescent squaraine derivative is positively correlated with that of the AChE inhibitor in solution. This new designed sensing system shows an obviously improved sensitivity toward target with a detection limit of 5 pg mL^–1 (0.018 nM) for the AChE inhibitor, comparing favorably with previously reported fluorometric methods. To our best knowledge, this new method is the first example of fluorometric enzymatic assay for AChE inhibitors based on such a signal-on principle and using a specific reaction, which has potential to offer an effective strategy for the detection of AChE inhibitors