Chan Cao

A Stimuli-Responsive Nanopore Based on a Photoresponsive Host-Guest System

The open-close states of the ion channels in a living system are regulated by multiple stimuli such as ligand, pH, potential and light. Functionalizing natural channels by using synthetic chemistry would provide biological nanopores with novel properties and applications. Here we use para-sulfonato-calix[4]arene-based host-guest supramolecular system to develop artificial gating mechanisms aiming at regulating wild-type α-HL commanded by both ligand and light stimuli. Using the gating property of α-hemolysin, we studied the host-guest interactions between para-sulfonato-calix[4]arene and 4, 4′-dipyridinium-azobenzene at the single-molecule level. Subsequently, we have extended the application of this gating system to the real-time study of light-induced molecular shuttle based on para-sulfonato-calix[4]arene and 4, 4′-dipyridinium-azobenzene at the single-molecule level. These experiments provide a more efficient method to develop a general tool to analyze the individual motions of supramolecular systems by using commercially available α-HL nanopores.

Analysis of a Single α-Synuclein Fibrillation by the Interaction with a Protein Nanopore

The formation of an α-synuclein fibril is critical in the pathogenesis of Parkinsons disease. The native unfolded α-synuclein monomer will translocate through an α-hemolysin nanopore by applied potential at physiological conditions in vitro. Applying a potential transformed α-synuclein into a partially folded intermediate, which was monitored by capture inside the vestibule of an α-hemolysin nanopore with a capture current of 20 ± 1.0 pA. The procedure involves the critical early stage of α-synuclein structural transformation. Further elongation of the intermediate produces a block current to 5 ± 0.5 pA. It is revealed that the early stage fibril of α-synuclein inside the nanopore is affected by intrapeptide electrostatic interaction. In addition, trehalose cleared the fibrillation by changing the surface hydrophobic interaction of A53T α-synuclein, which did not show any inhibition effect from WT α-synuclein. The results proved that the interpeptide hydrophobic interactions in the elongation of A53T α-synuclein protofilaments can be greatly weakened by trehalose. This suggests that trehalose inhibits the interpeptide interaction involved in protein secondary structure. The hydrophobic and electrostatic interactions are associated with an increase in α-synuclein fibrillation propensity. This work provides unique insights into the earliest steps of the α-synuclein aggregation pathway and provides the potential basis for the development of drugs that can prevent α-synuclein aggregation at the initial stage.

Accurate Data Process for Nanopore Analysis

Data analysis for nanopore experiments remains a fundamental and technological challenge because of the large data volume, the presence of unavoidable noise, and the filtering effect. Here, we present an accurate and robust data process that recognizes the current blockades and enables evaluation of the dwell time and current amplitude through a novel second-order-differential-based calibration method and an integration method, respectively. We applied the developed data process to analyze both generated blockages and experimental data. Compared to the results obtained using the conventional method, those obtained using the new method provided a significant increase in the accuracy of nanopore measurements.

Driven Translocation of Polynucleotides Through an Aerolysin Nanopore

Aerolysin has been used as a biological nanopore for studying peptides, proteins, and oligosaccharides in the past two decades. Here, we report that wild-type aerolysin could be utilized for polynucleotide analysis. Driven a short polynucleotide of four nucleotides length through aerolysin occludes nearly 50% amplitude of the open pore current. Furthermore, the result of total internal reflection fluorescence measurement provides direct evidence for the driven translocation of single polynucleotide through aerolysin.

Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition

Detection of DNA methylation in real human serum is of great importance to push the development of clinical research and early diagnosis of human diseases. Herein, taking advantage of stable pore structure of aerolysin in a harsh environment, we distinguish methylated cytosine from cytosine using aerolysin nanopore in human serum. Since wild-type (WT) aerolysin enables high sensitivity detection of DNA, the subtle difference between methylated cytosine and cytosine could be measured directly without any specific designs. Methylated cytosine induced a population of I/I0 = 0.53 while cytosine was focused on I/I0 = 0.56. The dwell time of methylated cytosine (5.3 ± 0.1 ms) was much longer than that of cytosine (3.9 ± 0.1 ms), which improves the accuracy for the discrimination of the two oligomers. Moreover, the pore-membrane system could remain stable for more than 2 h and achieve the detection of methylated cytosine with zero-background signal in the presence of serum. Additionally, event frequency of methylated cytosine is in correspondence with the relative concentration and facilitate the quantification of methylation.

Construction of an aerolysin nanopore in a lipid bilayer for single-oligonucleotide analysis

Nanopore techniques offer the possibility to study biomolecules at the single-molecule level in a low-cost, label-free and high-throughput manner. By analyzing the level, duration and frequency of ionic current blockades, information regarding the structural conformation, mass, length and concentration of single molecules can be obtained in physiological conditions. Aerolysin monomers assemble into small pores that provide a confined space for effective electrochemical control of a single molecule interacting with the pore, which significantly improves the temporal resolution of this technique. In comparison with other reported protein nanopores, aerolysin maintains its functional stability in a wide range of pH conditions, which allows for the direct discrimination of oligonucleotides between 2 and 10 nt in length and the monitoring of the stepwise cleavage of oligonucleotides by exonuclease I (Exo I) in real time. This protocol describes the process of activating proaerolysin using immobilized trypsin to obtain the aerolysin monomer, the construction of a lipid membrane and the insertion of an individual aerolysin nanopore into this membrane. A step-by-step description is provided of how to perform single-oligonucleotide analyses and how to process the acquired data. The total time required for this protocol is ∼3 d.

Real-time monitoring of the oxidative response of a membrane–channel biomimetic system to free radicals

A novel method for real-time monitoring of the oxidative response of a membrane-channel biomimetic system (MCBS) to free radicals is developed and the deduction of the buffering effect of MCBS is discussed.

Enhanced resolution of low molecular weight poly(ethylene glycol) in nanopore analysis.

A design with conjugation of DNA hairpin structure to the poly(ethylene glycol) molecule was presented to enhance the temporal resolution of low molecular weight poly(ethylene glycol) in nanopore studies. By the virtue of this design, detection of an individual PEG with molecular weight as low as 140 Da was achieved at the single-molecule level in solution, which provides a novel strategy for characterization of an individual small molecule within a nanopore. Furthermore, we found that the current duration time of poly(ethylene glycol) was scaled with the relative molecular weight, which has a potential application in single-molecule detection.

Direct Readout of Single Nucleobase Variations in an Oligonucleotide

Direct, low-cost, label-free, and enzyme-free identification of single nucleobase is a great challenge for genomic studies. Here, this study reports that wild-type aerolysin can directly identify the difference of four types of single nucleobase (adenine, thymine, cytosine, and guanine) in a free DNA oligomer while avoiding the operations of additional DNA immobilization, adapter incorporation, and the use of the processing enzyme. The nanoconfined space of aerolysin enables DNA molecules to be limited in the narrow pore. Moreover, aerolysin exhibits an unexpected capability of detecting DNA oligomers at the femtomolar concentration. In the future, by virtue of the high sensitivity of aerolysin and its high capture ability for DNA oligomers, aerolysin will play an important role in the studies of single nucleobase variations and open up new avenues for a broad range of nucleic-acid-based sensing and disease diagnosis.

Reply to Comment on Accurate Data Process for Nanopore Analysis

Anal. Chem. 2015, 87, 907−913. DOI: 10.1021/ac5028758 H we carefully consider and reply to the comments raised by Dr. Dunbar regarding the accuracy of our presented data process for nanopore analysis. Our work focuses on current blockage location by improving the accuracy of local threshold approach and the evaluation of current amplitude by applying an integration method. In this response, we provide a more detailed description of second-order-differential-based calibration (DBC) method. Moreover, the advantages of our proposed method have been discussed by comparing with conventional methods. Thanks to the simulation method provided in the comment, we further built a calibration method to decrease the errors in evaluation of short blockages in nanopore studies. Our improved methods are useful for the problem of extracting information from blockages which are faster than the instrument response. The pulse train model is a simplified model for processing the blockage current acquired in experiments. We agree with the opinions from Dr. Dunbar that the current model should be carefully used in a part of nanopore signal analysis, where the original shape of current blockage may appear to be ramplike. Therefore, we considered the pulse train model and data process based on this model is not appropriate for all of the nanopore experimental results. It can be used in the experiments where the transition between open pore state and blockage state is faster than the instrument response (up to 100 kHz bandwidth at current stage). In our method, the DBC method is developed to precisely locate the region of blockage, which largely eliminates the effect of random noise. Then, we adopt the criterion from Rant’s study to evaluate dwell time. It should be noted that the DBC method is not limited to the applications which are based on Rant’s criterion. DBC method could be incorporated into many other criterions of dwell time. As kindly proved in the comment, the unfiltered blockage and its filtered version have equal areas. In other words, the integrations of filtered blockage currents are hardly affected by the low-pass filter. Therefore, we adopted integrated area in calculating the current amplitude from the attenuated events. We appreciated that Dr. Dunbar provided good references and software for nanopore data analysis. The present methods for nanopore data analysis were listed but not limited in Table 1. The OpenNanopore software extracts multilevel information in the blockages from the solid-state nanopore by using the cumulative sums algorithm. The attenuations on the blockages induced by the low-pass filter are not a major concern in this software. For locating the blockage in the data process, the OpenNanopore uses localized thresholds method. In contrast, the DBC method eliminates the errors originating from the setting of thresholds for the blockages with unavoidable noise at two edges and the integrations of blockage amplitude we proposed are independent of the bandwidth. The QUB, another software suggested in the comment, is a professional software for single channel data analysis. It aims to simulate kinetics of single molecule and owns the advanced features in statistical analysis based on Hidden Markov model and K-mean algorithm. The studies mentioned in the comment (refs 10−12 in the comment) about the Hidden Markov model were not used in analyzing real data for blockage detection or did not take the influence of the filter into consideration. However, our methods exhibit good performances in analyzing real data from α-hemolysin nanopore experiments. Thus, our method has the advantage among the present process for nanopore data analysis. Prior to the implementation of the DBC method, we applied a Fourier series to fit the experimental blockages, which showed a good performance. Note that the role of fitting process is only to smooth the signal data. The fitted function is not based on the circuit model of the system. Therefore, the fitted parameters are not relevant to the frequency response of the system. Here, we listed the fitted parameters of the 4th-order Fourier series which are asked for in the comment (Table 2). As a kind reminder, we noticed that the description of the conventional method for evaluating the dwell time might lead to a misunderstanding. In our paper, the conventional method uses Ps2 to Pe4 in the measurement of dwell time, while the DBC method uses Ps3 to Pe4 in the measurement of dwell time. Briefly, both the conventional and DBC method chose the same stop point as Pe4 but use the different start points. Particularly, the comment raised a concern about the “criterion” of the conventional method used in the comparison of dwell time. The dwell time definition of Ps2 and Pe4 is a conventional method originating from Rant’s study. The points of Ps2 and Pe2 are located by using a threshold and the tracking back routine, which is similar to the two-threshold method. Both of these two methods are widely used in automatic data processes for searching the start and stop point of the blockage in nanopore analysis. However, the filter would affect the dwell time of blockages. Rant’s group proposed a modified criterion to measure the dwell time of blockage by choosing “the last (or only) local minimum of the pulse before the signal starts to return” as the stop point to cut down the overestimation of dwell time. Here we adopted their definitions as a conventional one. Therefore, the stop point is regarded as Pe4 for defining the conventional dwell time. The start point in their criterion is located as the last data point before the current drops below the baseline, Comment