High-temperature thermal disturbance elimination method based on multi-channel extraction and correction.

Digital image correlation (DIC) technology has been widely used in high-temperature measurement fields. However, due to the complexity of high-temperature environments, there are many interference factors that limit the development of high-temperature DIC technology, among which thermal disturbance is one of the most significant factors that severely affects the measurement accuracy of high-temperature DIC. In this paper, a multi-channel separation technique combined with a low-cost laser speckle device is proposed to eliminate thermal disturbance errors in high-temperature DIC measurements. First, a blue laser speckle generation system is independently designed to produce the most suitable speckle particles, and the best laser speckle is determined and projected onto the blue background white spot pattern. Then a green LED illuminates the sample to provide illumination for the sample's own grayscale characteristics. A color camera collects photos, and the obtained images are processed with channel separation to extract and calculate the displacement of different channels. Finally, the displacement fields of the green and blue channels are subtracted to separate the thermal disturbance error and correct the measurement values. In this paper, a laser speckle projection system is first assembled, followed by a comprehensive evaluation of the projected speckle and, finally, a DIC experimental system is constructed for verification experiments at both room temperature and high temperature, and the corrected values are compared with the true values. The results show that the corrected values are highly consistent with the true values, which verifies the reliability of the proposed method.

Highly sensitive α-hemolysin nanopore detection of MUC1 based on 3D DNA walker.

Mucin 1(MUC1) is an effective marker of breast cancer, so it is of great significance to develop a simple, sensitive and highly selective MUC1 detection sensor. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of MUC1. The presence of MUC1 triggered the modification of the DNAzyme walking chain on the surface of Fe3O4 nanoparticles and separation from the aptamer. In the presence of Zn2+, DNAzyme catalyzed hydrolytic cleavage of the hairpin substrate at the scissile rA. The DNAzyme was divided into two fragments and ssDNA was released. ssDNA products from the hairpin substrate can generate a current blocking signal during α-hemolysin nanopore testing. The frequency of signature events showed a linear response toward the concentration of MUC1 in the range of 0.01 nM-100 nM. The sensing system also exhibited high selectivity against other protein and can be used for the detection of real sample.

Simultaneous Dual-Site Identification of 5mC/8oG in DNA Triplex Using a Nanopore Sensor.

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5mC with 8oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5mC with 8oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5mC with 8oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.

Nanopore detects γ-radiation inhibited HIV-1 protease activity.

Inhibition of HIV-1 protease (PR) activity is realized by exposure to 60Co γ-radiation. The radiation effects on enzyme kinetics of HIV-1 PR are subsequently monitored using nanopore sensor. Substantial loss of proteolytic efficiency towards GagPol polypeptide is observed due to the radiation treatment. Results shows ~50% of GagPol polypeptide was not involved in HIV-1 PR proteolysis by exposure to ultra-low intensity of γ-radiation (0.1K Gy), and the values reach to over 90% with high γ-ray treatment. Besides, the inactivation effect is also verified in blood samples which contain the virus protease. Our finding provides the potential benefits of γ-radiation to inactivate viral proteinic function, and might be a complementary to the designation of HIV-1 PR inhibitors.

Enzyme Hinders HIV-1 Tat Viral Transport and Real-Time Measured with Nanopores.

HIV-1 Tat protein, an intercellular transporter with a determinant function of delivering "information-rich" molecules in viral multiplication, was tryptic-hydrolyzed and real-time single molecule-monitored in a transmembrane pore. The electrokinetic studies revealed the catalytic and inhibitory effects on enzymatic digestion associated with Ca2+ and Cu2+ ions, respectively, in response to binding interactions with trypsin. Our strategy permits accurate and distinguishable sensing of Ca2+ and Cu2+via an enzyme assay. In addition, considering the closer mimic of the real situation of HIV spread, measurements in the serum and on cells were also investigated. Transmembrane current measurements together with fluorescence microscopy imaging indicated the potential to perturb the Tat transport in the serum environment and on cells. Because the involved Tat proteolysis should prevent the occurrence of viral delivery, the presented method probably enables efficient hindrance to HIV-1 infection, in complementary to current traditional treatments.

Nanopore Technology for the Application of Protein Detection.

Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.

Reduction chemistry-assisted nanopore determination method for immunoglobulin isotypes.

Immunoglobulins can bind to an unlimited array of foreign antigens presented to the immune system. Among those isotypes, IgG and IgM play crucial roles in initial immune defense associated with innate immunity factors. Hence, the determination of IgG and IgM deficiencies or varying concentrations is widely used as a diagnostic indicator for immune deficiency disorders. Herein, we report a reduction chemistry-assisted nanopore method for IgG and IgM determination. TCEP (tris(2-carboxyethyl)phosphine) was used to cleave Ig proteins in fragments by means of disulfide bond reduction under different experimental conditions. This strategy enabled the observation of distinguishable current signals afforded by separated polypeptide fragments in an αHL nanopore. Together with molecular dynamics (MD) simulation results, highly effective electrostatic potentials and H-bonds, the dominant factors for these current signals, facilitated the capture of Ig fragments in an α-HL nanopore. More importantly, the signature signals were applicable for differentiating between IgG and IgM in blood serum without any problems of protein adsorption and clogging in the nanopore sensing. Furthermore, with comparative sensing sensitivity and selectivity, it is concluded that our method is a label-free single-molecule approach to measuring disease states that present as a result of the absence or over presence of immunoglobulin isotypes.