Investigation of the Interaction between a Bivalent Aptamer and Thrombin by AFM

Aptamers are a new class of molecular probes for protein recognition, detection, and inhibition. Multivalent aptamer–protein binding through aptamer assembly has been currently developed as an effective way to achieve higher protein affinity and selectivity. In this study, the specific interaction between bivalent aptamer Bi-8S and thrombin has been measured directly and quantitatively by atomic force microscopy to investigate the unbinding dynamics and dissociation energy landscape of the multivalent interaction. Bivalent aptamer Bi-8S contains thrombin’s two aptamers, 15apt and 27apt, which are linked by eight spacer phosphoramidites. The results revealed the sequential dissociation of the two aptamers. Moreover, the dynamic force spectroscopy data revealed that the 27apt’s binding to the thrombin remains largely unaffected by the eight-spacer phosphoramidites within Bi-8S. In contrast, the eight-spacer phosphoramidites stabilized the 15apt–thrombin binding

Potential Modulation on Total Internal Reflection Ellipsometry

Electrochemical-total internal reflection ellipsometry (EC-TIRE) has been proposed as a technique to observe the redox reactions on the electrode surface due to its high phase sensitivity to the electrolyte/electrode interface. In this paper, we mainly focus on the influence of the potential modulation on the TIRE response. The analysis suggests that both dielectric constant variation of gold and the electric double layer transformation would modulate the reflection polarization of the surface. For a nonfaradaic process, the signal of TIRE would be proportional to the potential modulation. To testify the analysis, linear sweep voltammetry and open circuit measurement have been performed. The results strongly support the system analysis

Label-Free Sandwich Imaging Ellipsometry Immunosensor for Serological Detection of Procalcitonin

Analysis of trace low molecular weight (LMW) proteins in serum using the label-free imaging ellipsometry (IE) immunosensor is still a challenge due to the lack of an effective signal amplification strategy and the serious nonspecific adsorption. Herein we have developed a sandwich strategy-mediated IE (SSIE) immunosensor to enable the immunodetection of LMW protein biomarkers in serum samples. We have first found that the weak binding affinity and the insufficient surface amount density of the ligand are two important factors which hinder the detection of LMW proteins in serum using the IE immunosensor. Then we have deduced that the sandwich strategy can amplify the detection signal of IE and avoid the nonspecific adsorption in serum. As a validation of the serological detection of LMW proteins, the SSIE immunosensor has been used to accomplish the quantitative detection of procalcitonin (PCT) in serum. Compared with other PCT analysis methodologies, the SSIE immunosensor enjoys the advantages of simplicity, rapidity, and sufficient sensitivity. Furthermore, we have proposed the criteria to predict the ability of the SSIE immunosensor for the detection of LMW protein biomarkers in serum, which can make the detection of LMW proteins smart and efficient

Antibody Oriented Immobilization on Gold using the Reaction between Carbon Disulfide and Amine Groups and Its Application in Immunosensing

Carbon disulfide (CS₂) can spontaneously react with amine groups to form dithiocarbamates on gold surface, providing the possibility to immobilize some compounds with primary or secondary amine groups in one step. Using this principle, an immunosensor interface prepared for immunoglobulin G (IgG) sensing surface toward anti-IgG has been fabricated for the first time by simply immersing gold slides into a mixed aqueous solution of CS₂ and protein A, followed by incubation in immunoglobulin G solution. The reaction between CS₂ and protein A has been followed by UV–vis spectroscopy, whereas cyclic voltammetry has been employed in the characterization of the modified gold surface with CS₂ and protein A, both methods indicating that protein A immobilization is implemented by CS₂. Conventional ellipsometry, atomic force microscopy (AFM), as well as surface plasmon resonance (SPR) have been used to evaluate the specific binding of protein A with IgG and IgG with anti-IgG, revealing that IgG is specifically captured to form the biosensing interface, maintaining its bioactivity. Compared to direct adsorption of IgG on the gold surface, the IgG sensing surface constructed of CS₂ and protein A is far more sensitive to capture anti-IgG as its target molecule. In addition, the modified surface is proven to have good capability to inhibit nonspecific adsorption, as supported by control experiments using lysozyme and BSA. To conclude, antibody immobilization using this one-step method has potential as a simple and convenient surface modification approach for immunosensor development

Image_1_Comparative study on the microbiota of colostrum and nipple skin from lactating mothers separated from their newborn at birth in China.TIF

Increasing studies have found breast milk (BM) contains its own microbiota. However, the route through which microbes enter the BM is still unclear. In order to verify the entero-mammary pathway of BM, we designed a rigorous study that prevented oral bacteria from contaminating the breast and nipple skin (NS) during baby nursing. Thirty-one healthy, postpartum mothers living in southern China who were immediately separated from their newborn after delivery were enrolled in this study. Using an aseptic protocol for sampling, sterile water was used to wash the NS and was then collected. Then the first drop of BM was discarded and colostrum was collected manually. Amplicon sequencing was performed targeting the V3–V4 region of the bacterial 16S rRNA gene, and the differences between the microbiota of the colostrum and NS were analyzed. Additionally, the effects of environmental factors, such as the delivery mode and intrapartum antibiotic exposure, on the diversity of the colostrum microbiota were also analyzed. We found significant differences in the α diversity and richness between the BM and NS as evidenced by richness, Chao1, and Simpson indices. There were 170 operational taxonomic units (OTUs) shared by colostrum and NS, while 111 and 87 OTUs were unique, respectively, as well as a clear distinction in OTUs was observed by unifrac binary analysis between them. Linear discriminant analysis effect size analysis found that anaerobes, such as Bifidobacterium and Pantoea at the genus level and enterobacteria including Enterobacteriaceae at the family level, were predominant in the colostrum, while the predominant bacteria on the NS were Bacteroides, Staphylococcus, and Parabacteroides at the genus level. BM is colonized by bacteria prior to baby suckling, and the diversity of the colostrum microbiota differs from that of the NS. The predominant microbiota taxa in BM indicated that they were likely to be transferred to the breast through the intestinal tract. Our study provides direct evidence for the revolutionary active migration hypothesis. Additionally, factors like intrapartum antibiotic exposure did not significantly affect the diversity of the microbiota in the BM. Therefore, it is suggested that mothers continue to provide BM for their newborns during separation.</p

Calibration curve and sensitivity of BIE for detecting CMV antibodies.

<p>(A) Grayscale images of different CMV antibody concentration levels detected; (B) 3-D grayscale distribution map of different concentrations of CMV antibodies detected; and (C) standard curve of CMV-3A as ligand to detect five serial dilutions of containing CMV antibodies (0.011, 0.043, 0.170, 0.681, and 2.725 IU/mL). In the first step, CMV-3A was immobilized as the ligand on two columns. In the second step, PBST buffer was added as a blank control to two areas on the first row. Simultaneously, purified CMV antibody was added as a positive control to two areas on the second row. Negative serum was added as a negative control to two areas on the third row. The serial dilutions of CMV antibodies were added as analytical samples on the following rows. The same concentration was measured in two duplicate areas.</p

Specificity and qualitative detection of CMV antibodies using BIE.

<p>(A) Grayscale images of different CMV antibodies samples; and (B) 3-D grayscale distribution map of different CMV antibody samples in image A. In the first step, CMV-3A was immobilized as the ligand on two columns. In the second step, PBST buffer was added as a blank control to two areas on the first row. Simultaneously, purified CMV antibody was added as a positive control to two areas on the second row. Normal serum without CMV antibodies was added as a negative control to two areas on the third row. According to ELISA data, No. 956 had a high CMV antibody concentration, and No. 933 and 978 had medium CMV antibody concentrations. To observe qualitative detection of CMV antibodies in serum, samples with higher concentrations were chosen.</p

Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry

<div><p>Background</p><p>Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices.</p><p>Methods</p><p>An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A.</p><p>Results</p><p>Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL.</p><p>Conclusions</p><p>This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening.</p></div

Detection of CMV antibodies in patient serum using BIE.

<p>In the first step, CMV-3A was immobilized as the ligand on six columns. In the second step, PBST buffer was added as a blank control to six areas on the first row. Simultaneously, purified CMV antibody was added as a positive control to the last two areas on the second row. Patient serum samples were added as analytical samples on the following areas, respectively. The same serum sample was measured in two duplicate areas (No.940, 959,938 no sample, P15-9, and PBST control are underlined).</p

Comparison of BIE with ELISA.

<p>P15-1 and P15-2 were used as healthy controls. Concentration of CMV antibody (0.5 IU/mL) located in the normal reference range (0.4–0.6 IU/mL). The correlation coefficient (<i>r</i>-value) and <i>P</i>-value were calculated.</p