Electrical Signal Reporter, Pore-forming Protein ... - ACS Publications

Subscriber access provided by the Henry Madden Library | California State University, Fresno

Article

Electrical Signal Reporter, Pore-forming Protein, for Rapid, Miniaturized, and Universal Identification of Microorganisms Yi Wan, Fengge Song, Guoqing Wang, Hong Liu, Meng An, Aimin Wang, Xi Wu, Chunxin Ma, and Ning Wang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Electrical Signal Reporter, Pore-forming Protein, for Rapid, Miniaturized, and Universal Identification of Microorganisms Yi Wana,b, Fengge Songa,b, Guoqing Wangc, Hong Liua,b, Meng Ana,b, Aimin Wanga,b, Xi Wud, Chunxin Maa, Ning Wanga* a

State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, No. 58, Renmin Avenue, Haikou, Hainan Province, 570228 b Marine Colleage, Hainan University, No. 58, Renmin Avenue, Haikou, Hainan Province, 570228 c College of Materials and Chemical Engineering, Hainan University No. 58, Renmin Avenue, Haikou, Hainan Province, 570228 d Shenzhen Institute for Drug Control, No.28, Gaoxinzhong 2nd Road, Shenzhen, Guangdong Province, China, 518057 ABSTRACT: Despite of recent advances in signal reporter-based assays for bacteria detection and profiling, the low-cost, ultrasensitive, accurate and fast diagnosis remains a challenge for better patient care. Herein, we present a novel bacteria identification method based on α-hemolysin-labelled sandwich assay (HLSA). A pore-forming protein α-hemolysin, is used as an electrical signal reporter. The assay takes advantage of the specific binding of target nucleic acid with two hybridization probes: capture probe decorated magnetic microparticles, and oligonucleotides detecting probe and α-hemolysin modified gold nanoparticles. αhemolysin was then released by competitive gold binding peptide incubation into an electric cell with a lipid bilayer between the electrodes. The nanopores formed by α-hemolysin on the lipid layer allowed target nucleic acid concentration dependent currents for quantification. Sandwich probes against 16S rRNAs of 10 common bacteria pathogens were designed and single cell level nucleic acid concentration detection was achieved. Compared with nanopore technique based DNA sequencing, HLSA gives a quantitive and straightforward readout that’s not dependent on ultrasensitive and expensive instrument (Axopatch 200B amplifier), thus is faster and requires no large-scale instruments. Also, since α-hemolysin modified nanoparticles will be washed out before the α-hemolysin releasing step without the target nucleic acid, no current will be detected and thus, the assay is more specific. The current strategy based on the electrical signal reporter offers a new insight for pathogen and virus diagnostics.

Bacterial contamination caused disease accounts for approximately one-third of global deaths and results in more than 2.0 billion illnesses each year1. In order to select the best treatment option for these patients, numerous traditional culture techniques for pathogenic bacteria identification have been developed, each varying in time, cost, sensitivity, specificity, and efficacy. However, those teniques are usually time-consuming and cannot identify some non-culturable bacteria2. Alternatively, in the last two decades, many other fast identification methods have beendeveloped including quantitative real-time PCR (qPCR)3, digital PCR4, and deep sequencing5. Although these methods above have higher sensitivity and can be faster than traditional culture techniques, they are generally too expensive to be applied in many poverty-stricken areas of the world.

In order to achieve cost-effective and highly sensitive target detection, several new methods based on signal reporters have been explored in the last few years. These signal reporters can improve the signal-to-noise ratio, decrease analysis time and allow simultaneous multiplex sample detection. For example, some proteins and functionalized nanocomplexes have been utilized as ultrasensitive signal reporters for the detection of targets based on different kinds of signals, including optical (Cas13a6, engineered T7 phage7, carbon dots8 and fluorescent protein9), nuclear magnetic (magneto-DNA nanoparticle system10 and hyperpolarized 129Xe11), colorimetric (invertase12 and catalase13), electrochemical ([Ru(NH3)6]Cl314,15 and methylene blue16), odorant (tryptophanase17 and lipase18) and acoustic (gas vesicles19,20) based reporters. For example, bacterial gene expression of acoustic reporter encoding gas vesicles allows the localized visualization of Escherichia coli

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. HLSA for the detection of bacterial 16S rRNA. The amplified product from bacterial 16S rRNA is then captured and collected by MBs probe (Step 1b), before hybridizing with HPG probe (Step 1a) to form a magnetic sandwich complex (Step 2). After the sandwich nanocomplexes were magnetically clustered, GBP-tagged α-hemolysins could be replaced from the nanocomplexes by adding excess GBP (Step3) and subsequently assembled in chamber cell (Step 4) and analysed using an eONE system (Step 5).

in gastrointestinal model and Salmonella typhimurium in tumour noninvasively at volumetric densities below 0.01% with high spatial resolution (less than 100 µm)20. Another example, Gootenberg et al. used Cas13a-mediated collateral cleavage with recombinase polymerase amplification to distinguish pathogenic bacteria with attomolar sensitivity and single-base mismatch specificity6. While most of these approaches above indicated ultrasensitive diagnosis, it still remains a significant challenge to integrate both high sensitivity and portable capabilities into one reporter-based system for bacteria detections. Here we have developed a new, general and low-cost strategy for the fast and highly-sensitive identification of pathogens without large-scale instrument based on α-hemolysin reporter mediated electrical signal. α-hemolysin is a protein toxin produced by Staphylococcus aureus that can cause red blood cells lysis through inserting into the cell membrane21. Upon insertion, α-hemolysin forms water-filled mushroom-like nanochannel oligomers22, which have been used to produce a nanopore on phospholipid bilayers for DNA sequencing23. In order to distinguish subtle differences among nucleic acid bases, ultra low current changes generated in this method (below 1 pA) must be resolved. Thus, relatively expensive low-noise data acquisition system and ultrasensitive patch clamp recording amplifer system are required for accurate detection. Inspired by the direct correlation between current

intensity induced nanopore and the concentration of αhemolysin, we have explored a complementary technique: the α-hemolysin-labelled sandwich assay (HLSA), for bacterial detection and phenotyping. The current method relies on a sandwich hybridization technique with two DNA probes and α-hemolysin as a signal reporter. The target nucleic acid was captured via the complimentary DNA probe on magnetic beads (MBs) followed by recruitment of α-hemolysin and detection probe dually modified gold nanoparticles (HPG) via the other DNA probe, which was also modified with engineered α-hemolysin, forming a sandwich nanocomplex. After a washing step, α-hemolysin was released from HPG in the sandwich nanocomplex by competitive replacement of gold binding peptide (GBP) and then self-assembled into nanopores on a lipid bilayer. The target concentration was determined by electrophysiology measurement of the direct current across the lipid bilayer. With the assistance of the miniaturized one-channel amplifier, HLSA is able to phenotype some low-concentration bacteria from patient specimens othewise undetectable by standard culture method. Experimental Section Primer and probe design The 16S rRNA gene sequences of different bacterial genera were downloaded from NCBI database. 50–70 nucleotides were selected as target regions and affinity probes and primers


Page 2 of 8

Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 2. Preparation and fabrication of HPG nanoprobe. The schematic diagram (a), SDS-PAGE (b), hemolysis activity(c), binding capability (BC%) (d) for ten DNA conjugated GNP of α-hemolysin with gold-binding peptide (GBP) in N-terminus (I), M-terminus (II), and Cterminus (III). Ultraviolet-Visible absorption spectra (e) of GNP, DNA-conjugated GNP and HPG probe. By measuring the peak absorbance value and assuming an extinction coefficient of 8.78×108 M-1 cm-1 for 20 nm GNP, the concentration of GNP, DNA-conjugated GNP and HPG probe were calculated to be ~4.1 nM, ~3.5 nM and ~2.8 nM, respectively. A small red shift of 3-5 nm in UV-vis spectra was obtained for DNA-conjugated GNP and HPG probe, indicating that DNA and GBP-tagged hemolysin was successfully connected with GNP. Vibrio parahaemolyticus (VP), Listeria monocytogenes (LM), Proteus mirabilis (PM), Enterococcus faecalis (EF), Klebsiella pneumonia (KP), Streptococcus pyogenes (SP), Staphylococcus aureus (SA), Pseudomonas aeruginos (PA), Escherichia coli (EC), and Eberthella typhi (ET).

were designed using MegAlign software (DNASTAR)10. To analyze the 16S rRNA of different genus types, both specific probes (detection probe and capture detection with c.a. 20 nucleotides) were utilized to recognize to sequences within the target regions of interest. All oligonucleotides used for the primers and probes were offered by Sangon Biotech. Probe specificity was carried out by polyacrylamide gel electrophoresis24.

Magnetic conjugations 20 uL 5 µM Biotin-DNA in water was mixed with the 100 µl MBs solution (4 mg/ml, NewEnglands Biolabs) and incubated for 1 hour at room temperature. The magnetic beads complex was washed using PBS for six times. The number of oligonucleotides conjugated onto the MBs was quantified using the Quant-iT™ OliGreen® ssDNA Reagent (Invitrogen by Thermo Fisher Scientific, USA)25.

Expression and purification of GBP-tagged α-hemolysin: The cloning vector inserted with gold binding peptide sequence in C-terminus, N-terminus, or M-terminus was provided by NovoPro Bioscience Inc., China and the sequence confirmed by Sangon Biotech., China. Transformed Escherichia coli strain transtta DE3 (TransGen Biotech, China) using an expression vector (PET 22b(+)) was incubated at 37°C in LB medium supplemented with 50 µg mL−1 ampicillin with shaking at 150 rpm until reach the early stationary phase. To obtain the protein of interest, the culture with isopropyl-βd-thiogalactopyranoside (IPTG, 0.5 mM) was further shaked for 18 h at 25°C. Bacterial cells were obtained by centrifugation at 5000g for 10 min at 4°C, and then obstructed by sonication. The debris was dismantled by centrifugation at 12,000g for 30 min at 4°C, and supernatant was added onto Bio-Scale Mini Nuvia IMAC Ni-Charged (Bio-Rad Laborataries, Inc., USA). Fractions containing α-hemolysin were concentrated and further purified on Enrich SEC 650 High-Resolution Size Exclusion Columns (Bio-Rad Laborataries, Inc., USA). GBP-tagged hemolysin was confirmed by SDS-PAGE (Bio-Rad Laborataries, Inc., USA).

Synthesis of HPG probe 20 µL of 0.1 mM thiol-DNA in Millipore water, 2 µL of 1 M PBS at pH 5.5 and 2 µL of 30 mM Tris(2carboxyethyl)phosphine hydrochloride (Sigma-Aldrich) in water were mixed and reacted at room temperature for 1 hour. Then the freshly cleaved oligonucleotides were added into gold nanoparticles (1OD/1 mL) and were left shaking for 12 hours at room temperature. 2M NaCl was added into the reaction mix to bring the NaCl concentration to 0.1M and incubated for 2 hours. Small portions of 2M NaCl were added over 1 hour increments until a final concentration of 1.0 M was reached. The solution was kept at room temperature for overnight. To remove un-reacted thiol-DNA, the mixture was washed with buffer 4 times12. The number of oligonucleotides conjugated onto the GNP was quantified using the Quant-iT™ OliGreen® ssDNA Reagent (Invitrogen by Thermo Fisher Scientific, USA) according to recommended protocol. HPG probes were fabricated as follow: the DNA-conjugated GNP (2 nM, 0.2ml) were incubated with excess GBP-tagged hemolysin (1µM, 0.2ml) in 10 mM PBS for 4 hours at room temperature. The mixture was purified with Amicon Ultra-



Figure 3. Sensitivity and specificity detection using the HLSA and qPCR system. (a) Bacterial detection by HLSA assay. Samples with varying numbers of S. aureus were used. Probes specific for different bacteria were utilized for detecting various bacterial types. (105 cfu mL-1) (b). Heat maps comparing the specificity of the HLSA assay (c) with that of qPCR (d). Results are shown as mean+s.d. All specimens for the tests were measured in triplica.

100K for ten times to obtain HPG probe. The number of GBPtagged hemolysin conjugated onto the GNP was quantified using the Qubit™ Protein Assay Kit (Lot: 1790704, Molecular Probes by Life Technologies, USA). Bacteria culture and RNA extraction All bacteria were purchased from the China Center of Industrial Culture Collection (CICC). Bacteria were seeded and cultured in suspension using the following media: Escherichia coli (CICC 10412) in Luria-Bertani (LB) media; Eberthella typhi (CICC 21483), Pseudomonas aeruginosa (CICC 21625), Klebsiella pneumonia (CICC 10870) and Proteus mirabilis (CICC 21516) , Staphylococcus aureus (CICC 10384), Listeria monocytogenes (CICC 21633), Streptococcus pyogenes (CICC 10373), and Enterococcus faecalis (CICC 10396) in trypticase soy broth; Vibrio parahaemolyticus (CICC 21617) in trypticase soy broth containing 3.5% NaCl. Bacteria incubated at 37°C in medium with shaking at 150 rpm until reaching 5×108 cfu mL-1. For RNA extraction of Gram-negative bacteria with Bacterial RNA kit (Omega, USA), bacteria were first centrifuged (6,000 r.p.m., 10 min), and pellets were incubated with lysozyme. For RNA extraction of Gram-positive bacteria with Bacterial RNA kit, bacteria were first centrifuged (6,000 r.p.m., 10 min), and pellets were incubated with lysozyme containing ∼200 mg of zirconia beads. The bacterial

cells were beaten on a tissue cell-destroyer (NewZongKe Viral Disease Control Bio-Tech Ltd., China) with a small tube adapter for 10 min. After solvent extraction of the RNA followed by HiBind® RNA Mini Column and washing, the final RNA yield was measured using NanoDrop Microvolume Spectrophotometers (Thermo Scientific, USA) and agarose gel electrophoresis (DYY-6D, Beijing Liuyi Biotechnology Co. Ltd, China). HLSA experiments HLSA experiments were performed as follow: the solution containing 40 µl of MBs probes at 2 mg/mL and 40 µl of assay buffer (0.2 M NaCl, 10 mM PBS, 0.1% Tween 20, pH 7.4) were added into the sample solution (20 ul of bacteria-derived DNA samples), followed by incubation at 37 oC for 30 min. The MBs-target nanocomplexes formed were then separated magnetically and washed ten times with the assay buffer, followed by incubating with 40 µl of HPG probes (2.0 nM) at 37 oC for another 30 min. The sandwich nanocomplexes were again extracted magnetically and washed with the assay buffer for six times. GBP-labeled hemolysin was released from the nanocomplexes by adding 0.1ml of 2 µM GBP, followed by incubation for at least 20 min under gentle shaking at 37 oC. After centrifugation, the supernatant containing the released GBP-labeled hemolysin was added into the electrochemical


Page 4 of 8

Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry chambers, and then observed under Miniaturized One-Channel Amplifier (eONE, Elements SRL, Italy). For nanopore current measurement using miniaturized one-channel amplifier, 1.25 mg of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Inc., USA) was dissolved in 100 µl of decane to prepare a lipid solution with a concentration of 12.5 mg/ml. The lipid solution was painted on aperture (diameter=200 µm) of the Delrin perfusion cup (Warner Instruments, LLC, USA) and dried under a gentle air flow. Then, a positive potential (+100 mV) was applied and 10 µl of the supernatant containing the released GBP-labeled hemolysin from the nanocomplexes in the electrolyte solutions (1.0 M KCl, 10 mM Tris and 1.0 mM EDTA, pH 8.0). HLSA for clinic samples: This study was conducted according to the principles expressed in the Declaration of Helsinki. For clinic samples, samples were provided friendly from the 187th hospital of People's Liberation Army of China and analysed with the HLSA assay, before being compared to conventional culture and qPCR. After solvent extraction of the RNA, bacterial detection and identification were measured based on the above-mentioned NLSA experiments.

nucleic acid target and HPG were mixed and incubated for 30 min. Upon hybridization, the MBs were densely coated with hundreds of GNP as confirmed by TEM (Figure S11). After the sandwich nanocomplexes were magnetically clustered, GBP-tagged α-hemolysins were displaced from the nanocomplexes by adding excess GBP, as illustrated by SDSPAGE (Figure S5). Control experiments without target showed negligible GNP on the surface of MBs indicating excellent selectivity of the probes against target nucleic acid. The prospect applications of HLSA platform are fast detection and phenotyping of microorganisms including bacteria, viruses, and fungi29. To demonstrate the capability our approach, we chose S. aureus as the model organism. HLSA sensitivity was determined to be 1 pM for DNA target (Figure S6) and single cell level nucleic acid concentrationfor bacteria (Figure 3a and Figure S7) when combined with asymmetric PCR amplification and magnetic beads enrichment. These merits make HLSA an excellent diagnostic system for identification of bacteria.

Results and Discussion As show in Figure 1, in HLSA work flow, bacterial RNA was extracted using Bacterial RNA kit (Omega), and target regions within the 16S rRNA were then amplified by RT-PCR. The single-strand DNA generated was recognized and captured by the two probes (MBs and HPG) to form a sandwich. GBPlabeled α-hemolysin was released from the nanocomplexes by adding GBP due that GBP shows a strongly binding capability with GNP. After magnetic separation to remove the sandwich complex, the supernatant containing the released GBP-tagged α-hemolysin was added into the electrochemical chambers with a lipid bilayer (diameter = 4 nm) in between the two electrodes, and the current across the lipid membrane was measured. This approach features a constant current generated by the insertion of heptamer α-hemolysins on lipid membrane, which can last for more than ten minutes and is proportional to the target nucleic acid concentration. To assemble HPG probe, α-hemolysins was genetically encoded with a gold binding peptide (VSGSSPDS)26,27 at the C-terminus, M-terminus, and N-terminus (Figure 2, and S2). The M-terminus GBP-tagged α-hemolysin showed the highest hemolytic activity (Figure 2c) and the strongest GNP binding capacity (Figure 2d), thus was incubated with DNAconjugated GNP to generate HPG probe (47-151 GBP-tagged α-hemolysins for HPG in Table S2). The HPG probe was purified by ultracentrifugal filtration against assay buffer. The conjugation of GBP-tagged α-hemolysin and detection probe were characterized and confirmed by UV-Vis spectra (Figure 2e). A small red shift of 3-5 nm in UV-Vis spectra was detected for DNA-conjugated GNP and HPG, indicating that detection probe and GBP-tagged α-hemolysin was successfully installed on GNP28. The numbers of DNA probes per bead and GNP were determined to be 400,000–2000,000 and 30–110 (Table S2), respectively. Streptavidin magnetic beads (MB, 1 um in diameter, New England Biolabs) were incubated with biotinylated-capture DNA to form MB probe. For the sandwich assay, the capture probe-modified MBs,

Figure 4. Detection of bacteria by the HLSA system (a) and qPCR technique (b) using specific probes for each bacteria type in clinical samples. Clinical specimens (2 ml for each sample) were processed. One of the seven clinical specimens was negative, which confirmed with standard culture method. The other six specimens were positive. Results are shown as mean+s.d. All specimens for the tests were measured in triplicate.

The high degree of species-specific variable regions, V3, of the 16S rRNA gene has be used for bacterial phenotyping30-35. Here, we designed a panel of primers and probes for amplification and detection of specific target regions within different pathogens (Table S1 and Figure S1), including Escherichia coli, Eberthella typhi, Listeria monocytogenes, Vibrio parahaemolyticus, Streptococcus pyogenes,



Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumonia and Proteus mirabilis. Figure 3b and 3c represent the identification of above mentioned pathogen via HLSA by bacterial 16S rRNA amplification with background level response from other strains. HLSA completely genotyped each species and had low cross-reactivity as well as high selectivity with negligible offtarget binding. Remarkably, we also found that the specificity of the HLSA technique was superior to that of regular qPCR (Figure 4, Figure S8b, S9 and S10). The HLSA detection of pathogenic nucleic acid at ultralow level with a similar traditional sandwich assay could be a powerful substitute to costly qPCR analysis for diagnosing pathogenic infections. However, to execute the HLSA in resource-constrained areas, it is also indispensable to develop the robustness of the analytical protocols against possible impurities existed in actual matrices. We further explored whether the HLSA would be effective in clinical diagnostic applications that require high sensitivity and selectivity. A series of blood samples from six patients with suspected infections and healthy adult’s sample as a control were collected and assayed by conventional culture, qPCR as well as the HLSA(Table S3). Samples were repeatedly centrifuged and pellets were treated with the lysing buffer to remove impurities, before treating with bacterial RNA kit. HLSA was able to detect pathogenic-positive specimens that were undetectable by the standard culture assay (Table S3). Among these techniques, HLSA (assay time, 2-3 hours) has same levels of sensitivity to this of qPCR (assay time, 2 hours), the clinically approved ultrasensitive nucleic acid analysis technique, whereas conventional culture (assay time, 3–5 days) was not sensitive enough to measure low levels of bacterial target. In summary, despite of the complex components of patient samples, the HLSA approach can successfully identify pathogen from human sera with high fidelity(Figure 4). The results undoubtedly demonstrate that HLSA strategy is suitable for direct pathogen detection from patient samples. Conclusion In conclusion, we have developed a generally applicable and low-cost strategy for the fast and ultrasensitive identification of pathogens without any large-scale instrument. By measuring nanopore-mediated current signal, the quantification and identification of single cell level bacterial can be achieved simultaneously for the first time in only 2 hours. The current assay based on α-hemolysin as the electrical signal reporter for nucleic acid detection, can be easily applied to the detection of many other biologically important analytes. The relatively low cost and fast detection system holds great potential applications to control infectious diseases, especially in resource limited areas. Further optimization with automated microarray technology coupled with bioengineering and electrical engineering, can expand its current application to other areas for example, single-cell detection without PCR and early diagnosis of genetic disorders.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Experimental Procedures, Sequences of probes and primers for bacterial detection, Quantification of DNA probe conjugations on MBs and HPG, and Real-time PCR for differential detection.

AUTHOR INFORMATION Corresponding Author *[email protected] (Ning Wang)

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We gratefully acknowledge support from Hainan Provincial Natural Science Foundation of China (418QN206) and Research Foundation of Hainan Unversity (KYQD(ZR)1711).

REFERENCES (1) Havelaar, A. H.; Cawthorne, A.; Angulo, F.; Bellinger, D.; Corrigan, T.; Cravioto, A.; Gibb, H.; Hald, T.; Ehiri, J.; Kirk, M.; Lake, R.; Praet, N.; Speybroeck, N.; de Silva, N.; Stein, C.; Torgerson, P.; Kuchenmueller, T.; Epidemiology, F. D. B., WHO Initiative to Estimate the Global Burden of Foodborne Diseases. Lancet 2013, 381, 59-59. (2) Kilian, M.; Bülow, P., Rapid identification of Enterobacteriaceae. APMIS 1979, 87, 271-276. (3) Vliegen, I.; Jacobs, J. A.; Beuken, E.; Bruggeman, C. A.; Vink, C., Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene. J. Microbiol. Meth. 2006, 66, 156-164. (4) Luo, J.; Li, J. H.; Yang, H.; Yu, J. P.; Wei, H. P., Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR. J. Clin. Microbiol. 2017, 55, 2946-2955. (5) Xiang, L. X.; He, D.; Dong, W. R.; Zhang, Y. W.; Shao, J. Z., Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish. BMC Genomics 2010, 11. (6) Gootenberg, J. S.; Abudayyeh, O. O., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 2017, 356, 438-442. (7) Zhou, X.; Cao, P.; Zhu, Y.; Lu, W. G.; Gu, N.; Mao, C. B., Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish. Nat. Mater. 2015, 14, 1058-1064. (8) Zhu, S.; Meng, Q.; Wang, L.; Zhang, J.; Song, Y.; Jin, H.; Zhang, K.; Sun, H.; Wang, H.; Yang, B., Highly Photoluminescent Carbon Dots for Multicolor Patterning, Sensors, and Bioimaging. Angew. Chem. Inter. Ed. 2013, 52, 3953-3957. (9) Rana, S.; Le, N. D. B.; Mout, R.; Saha, K.; Tonga, G. Y.; Bain, R. E. S.; Miranda, O. R.; Rotello, C. M.; Rotello, V. M., A multichannel nanosensor for instantaneous readout of cancer drug mechanisms. Nat. Nanotechnol. 2014, 10, 65-69. (10) Chung, H. J.; Castro, C. M.; Im, H.; Lee, H.; Weissleder, R., A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria. Nat. Nanotechnol. 2013, 8, 369-375. (11) Palaniappan, K. K.; Ramirez, R. M.; Bajaj, V. S.; Wemmer, D. E.; Pines, A.; Francis, M. B., Molecular Imaging of Cancer Cells Using a Bacteriophage-Based 129Xe NMR Biosensor. Angew. Chem. Int. Ed. 2013, 52, 4849-4853.


Page 6 of 8

Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry (12) Xiang, Y.; Lu, Y., Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nat. Chem. 2011, 3, 697-703. (13) de la Rica, R.; Stevens, M. M., Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nat. Nanotechnol. 2012, 7, 821-824. (14) Das, J.; Cederquist, K. B.; Zaragoza, A. A.; Lee, P. E.; Sargent, E. H.; Kelley, S. O., An ultrasensitive universal detector based on neutralizer displacement. Nat. Chem. 2012, 4, 642. (15) Das, J.; Ivanov, I.; Montermini, L.; Rak, J.; Sargent, E. H.; Kelley, S. O., An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. Nat. Chem. 2015, 7, 569. (16) Hu, J.; Wang, T.; Kim, J.; Shannon, C.; Easley, C. J., Quantitation of Femtomolar Protein Levels via Direct Readout with the Electrochemical Proximity Assay. J. Am. Chem. Soc. 2012, 134, 7066-7072. (17) Xu, Y.; Zhang, Z.; Ali, M. M.; Sauder, J.; Deng, X.; Giang, K.; Aguirre, S. D.; Pelton, R.; Li, Y.; Filipe, C. D. M., Turning Tryptophanase into Odor-Generating Biosensors. Angew. Chem. Int. Ed. 2014, 53, 2620-2622. (18) Duncan, B.; Le, N. D. B.; Alexander, C.; Gupta, A.; Yesilbag Tonga, G.; Yazdani, M.; Landis, R. F.; Wang, L.-S.; Yan, B.; Burmaoglu, S.; Li, X.; Rotello, V. M., Sensing by Smell: Nanoparticle–Enzyme Sensors for Rapid and Sensitive Detection of Bacteria with Olfactory Output. ACS Nano 2017, 11, 53395343. (19) Shapiro, M. G.; Ramirez, R. M.; Sperling, L. J.; Sun, G.; Sun, J.; Pines, A.; Schaffer, D. V.; Bajaj, V. S., Genetically encoded reporters for hyperpolarized xenon magnetic resonance imaging. Nat. Chem. 2014, 6, 630-635. (20) Bourdeau, R. W.; Lee-Gosselin, A.; Lakshmanan, A.; Farhadi, A.; Kumar, S. R.; Nety, S. P.; Shapiro, M. G., Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 2018, 553, 86. (21) Mansouri, S.; Pahlavanzadeh, F., Hemolysin production, salt tolerance, antibacterial resistance, and prevalence of extended spectrum beta-lactamases in Proteus bacilli isolated from clinical and environmental sources. Jundishapur J. Microb. 2009, 2, 97104. (22) Chalmeau, J.; Monina, N.; Shin, J.; Vieu, C.; Noireaux, V., alpha-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression. BBA-Biomembranes 2011, 1808, 271-278. (23) Clarke, J.; Wu, H. C.; Jayasinghe, L.; Patel, A.; Reid, S.; Bayley, H., Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 2009, 4, 265-270. (24) Park, K. S.; Chung, H. J.; Khanam, F.; Lee, H.; Rashu, R.; Bhuiyan, M. T.; Berger, A.; Harris, J. B.; Calderwood, S. B.; Ryan, E. T.; Qadri, F.; Weissleder, R.; Charles, R. C., A magnetoDNA nanoparticle system for the rapid and sensitive diagnosis of enteric fever. Sci Rep-Uk 2016, 6, 32878. (25) Zhang, Y. X.; Wu, Q. P.; Wei, X. H.; Zhang, J. M.; Mo, S. P., DNA aptamer for use in a fluorescent assay for the shrimp allergen tropomyosin. Microchim. Acta 2017, 184, 633-639. (26) Li, Y.; Tang, Z. H.; Prasad, P. N.; Knecht, M. R.; Swihart, M. T., Peptide-mediated synthesis of gold nanoparticles: effects of peptide sequence and nature of binding on physicochemical properties. Nanoscale 2014, 6, 3165-3172. (27) Kim, J.; Kim, D. H.; Lee, S. J.; Rheem, Y.; Myung, N. V.; Hur, H. G., Synthesis of gold structures by gold-binding peptide governed by concentration of gold ion and peptide. Biosci. Biotech. Bioch. 2016, 80, 1478-1483. (28) Li, F.; Zhang, H. Q.; Dever, B.; Li, X. F.; Le, X. C., Thermal Stability of DNA Functionalized Gold Nanoparticles. Bioconjugate Chem. 2013, 24, 1790-1797.

(29) Cao, C.; Liao, D. F.; Yu, J.; Tian, H.; Long, Y. T., Construction of an aerolysin nanopore in a lipid bilayer for singleoligonucleotide analysis. Nat. Protoc. 2017, 12, 1901-1911. (30) Brown, C. T.; Hug, L. A.; Thomas, B. C.; Sharon, I.; Castelle, C. J.; Singh, A.; Wilkins, M. J.; Wrighton, K. C.; Williams, K. H.; Banfield, J. F., Unusual biology across a group comprising more than 15% of domain Bacteria. Nature 2015, 523, 208-U173. (31) Justesen, U. S.; Skov, M. N.; Knudsen, E.; Holt, H. M.; Sogaard, P.; Justesen, T., 16S rRNA Gene Sequencing in Routine Identification of Anaerobic Bacteria Isolated from Blood Cultures. J. Clin. Microbiol. 2010, 48, 946-948. (32) Teng, J. L. L.; Yeung, M. Y.; Yue, G.; Au-Yeung, R. K. H.; Yeung, E. Y. H.; Fung, A. M. Y.; Tse, H.; Yuen, K. Y.; Lau, S. K. P.; Woo, P. C. Y., In silico analysis of 16S rRNA gene sequencing based methods for identification of medically important aerobic Gram-negative bacteria. J. Med. Microbiol. 2011, 60, 1281-1286. (33) Poddar, A.; Lepcha, R. T.; Mukherjee, D.; Bhattacharyya, D.; Das, S. K., Comparative analysis of 16S rRNA signature sequences of the genus Idiomarina and Idiomarina woesei sp. nov., a novel marine bacterium isolated from the Andaman Sea. Res. Microbiol. 2014, 165, 501-507. (34) Han, L. Z.; Liu, Y. B.; He, A. G.; Zhao, D. G., 16S rRNA gene phylogeny and tfdA gene analysis of 2,4-D-degrading bacteria isolated in China. World J. Microb. Biot. 2014, 30, 25672576. (35) Hsieh, K. W.; Patterson, A. S.; Ferguson, B. S.; Plaxco, K. W.; Soh, H. T., Rapid, Sensitive, and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic Electrochemical Quantitative Loop-Mediated Isothermal Amplification. Angew. Chem. Int. Ed. 2012, 51, 4896-4900.




Page 8 of 8

Electrical Signal Reporter, Pore-forming Protein ... - ACS Publications

Recommend Documents