Development of an Integrated CMUTs-Based Resonant Biosensor for Label-Free Detection of DNA with Improved Selectivity by Ethylene-Glycol Alkanethiols

Gravimetric resonant-inspired biosensors have attracted increasing attention in industrial and point-ofcare applications,enabling label-free detection of biomarkers such as DNA and antibodies.Capacitive micromachined ultrasonic transducers(CMUTs)are promising tools for developing miniaturized highperformance biosensing complementary metal–oxide–silicon(CMOS)platforms.However,their operability is limited by inefficient functionalization,aggregation,crosstalk in the buffer,and the requirement for an external high-voltage(HV)power supply.In this study,we aimed to propose a CMUTs-based resonant biosensor integrated with a CMOS front–end interface coupled with ethylene–glycol alkanethiols to detect single-stranded DNA oligonucleotides with large specificity.The topography of the functionalized surface was characterized by energy-dispersive X-ray microanalysis.Improved selectivity for onchip hybridization was demonstrated by comparing complementary and non-complementary singlestranded DNA oligonucleotides using fluorescence imaging technology.The sensor array was further characterized using a five-element lumped equivalent model.The 4 mm^(2) application-specific integrated circuit chip was designed and developed through 0.18 lm HV bipolar-CMOS-double diffused metal–oxide–silicon(DMOS)technology(BCD)to generate on-chip 20 V HV boosting and to track feedback frequency under a standard 1.8 V supply,with a total power consumption of 3.8 mW in a continuous mode.The measured results indicated a detection sensitivity of 7.943×10^(-3) lmol·L^(-1)·Hz^(-1) over a concentration range of 1 to 100 lmol·L^(-1).In conclusion,the label-free biosensing of DNA under dry conditions was successfully demonstrated using a microfabricated CMUT array with a 2 MHz frequency on CMOS electronics with an internal HV supplier.Moreover,ethylene–glycol alkanethiols successfully deposited self-assembled monolayers on aluminum electrodes,which has never been attempted thus far on CMUTs,to enhance the selectivity of bio-functionalization.The findings of this study indicate the possibility of full-on-chip DNA biosensing with CMUTs.

A nano-metallic-particles-based CMOS image sensor for DNA detection

In this paper we report on a study of the CMOS image sensor detection of DNA based on self-assembled nano- metallic particles, which are selectively deposited on the surface of the passive image sensor. The nano-metallic particles effectively block the optical radiation in the visible spectrum of ordinary light source. When such a technical method is applied to DNA detection, the requirement for a special UV light source in the most popular fluorescence is eliminated. The DNA detection methodology is tested on a CMOS sensor chip fabricated using a standard 0.5 gm CMOS process. It is demonstrated that the approach is highly selective to detecting even a signal-base mismatched DNA target with an extremely-low-concentration DNA sample down to 10 pM under an ordinary light source.

Early detection of circulating tumor DNA and successful treatment with osimertinib in thr790met-positive leptomeningeal metastatic lung cancer:A case report

BACKGROUND Patients diagnosed with non-small-cell lung cancer with activated epidermal growth factor receptor mutations are more likely to develop leptomeningeal(LM)metastasis than other types of lung cancers and have a poor prognosis.Early diagnosis and effective treatment of leptomeningeal carcinoma can improve the prognosis.CASE SUMMARY A 55-year-old female with a progressive headache and vomiting for one month was admitted to Peking University First Hospital.She was diagnosed with lung adenocarcinoma with osseous metastasis 10 months prior to admittance.epidermal growth factor receptor(EGFR)mutation was detected by genomic examination,so she was first treated with gefitinib for 10 months before acquiring resistance.Cell-free cerebrospinal fluid(CSF)circulating tumor DNA detection by next-generation sequencing was conducted and indicated the EGFR-Thr790Met mutation,while biopsy and cytology from the patient’s CSF and the first enhanced cranial magnetic resonance imaging(MRI)showed no positive findings.A month later,the enhanced MRI showed linear leptomeningeal enhancement,and the cytology and biochemical examination in CSF remained negative.Therefore,osimertinib(80 mg/d)was initiated as a second-line treatment,resulting in a good response within a month.CONCLUSION This report suggests clinical benefit of osimertinib in LM patients with positive detection of the EGFR-Thr790Met mutation in CSF and proposes that the positive findings of CSF circulating tumor DNA as a liquid biopsy technology based on the detection of cancer-associated gene mutations may appear earlier than the imaging and CSF findings and may thus be helpful for therapy.Moreover,the routine screening of chest CT with the novel coronavirus may provide unexpected benefits。

Detection of DNA Bases Using Fe Atoms and Graphene

The adsorption of DNA bases on a magnetic probe composed of Fe atoms and graphene is studied by using first- principles calculations. The stability of geometry, the electronic structure and magnetic property are investigated. The results indicate that four DNA bases, i.e., adenine, thymine, cytosine and guanine, can all be adsorbed on the probe solidly. However, the magnetic moments of the composite structure can be observed only when adenine adsorbs on the probe. In the cases of the adsorption of the other three bases, the magnetic moments of the composite structure are zero. Based on the significant change of magnetic moment of the composite structure, adenine can be distinguished conveniently from thymine, cytosine and guanine. This work may provide a new way to detect DNA bases.

Silicon nanowire formed via shallow anisotropic etching Si-ash-trimming for specific DNA and electrochemical detection

A functionalized silicon nanowire field-effect transistor （SiNW FET） was fabricated to detect single molecules in the pM range to detect disease at the early stage with a sensitive, robust, and inexpensive method with the ability to provide specific and reliable data. The device was designed and fabricated by indented ash trimming via shallow anisotropic etching. The approach is a simple and low-cost technique that is compatible with the current commercial semiconductor standard CMOS process without an expensive deep reactive ion etcher. Specific electric changes were observed for DNA sensing when the nanowire surface was modified with a complementary captured DNA probe and target DNA through an organic linker （--OCH2CH3） using organofunctional alkoxysilanes （3-aminopropyl） triethoxysilane （APTES）. With this surface modification, a single specific target molecule can be detected. The simplicity of the sensing domain makes it feasible to miniaturize it for the development of a cancer detection kit, facilitating its use in both clinical and non-clinical environments to allow non-expert interpretation. With its novel electric response and potential for mass commercial fabrication, this biosensor can be developed to become a portable/point of care biosensor for both field and diagnostic applications.

Detection of DNA Mutations Using Novel SERS （Surface-Enhanced Raman Spectroscopy） Diagnostic Platform

This article describes the detection of DNA mutations using novel Au-Ag coated GaN substrate as SERS （surface-enhanced Raman spectroscopy） diagnostic platform. Oligonucleotide sequences corresponding to the BCR-ABL （breakpoint cluster region-Abelson） gene responsible for development of chronic myelogenous leukemia were used as a model system to demonstrate the discrimination between the wild type and Met244Val mutations. The thiolated ssDNA （single-strand DNA） was immobilized on the SERS-active surface and then hybridized to a labeled target sequence from solution. An intense SERS signal of the reporter molecule MGITC was detected from the complementary target due to formation of double helix. The SERS signal was either not observed, or decreased dramatically for a negative control sample consisting of labeled DNA that was not complementary to the DNA probe. The results indicate that our SERS substrate offers an opportunity for the development of novel diagnostic assays.

Comparison of Genomic DNA Extraction Methods for Chenopodium quinoa Willd

To rapidly obtain high-quality genomic DNA from Chenopodium quinoa Willd, the genomic DAN in different tissues （leaves, stems and roots） of Chenopodi- um quinoa Willd was extracted by modified CTAB method, SDS method and high- salt Iow-pH method, respectively. The quality and yield of extracted DNA was deter- mined using agarose gel electrophoresis and UV spectrophotometry. At the same time, the PCR-SSR and SSCP molecular detection was also performed. The results showed that the gel test strips, without obvious decomposition, of all the extraction methods were relatively obvious; the genomic DNA yield extracted by modified CTAB method was highest, followed by that by SDS method, and the genomic DNA extracted by high-salt Iow-pH method was lowest： the genomic DNA yields extracted by different methods from Chenopodium quinoa Wiltd leaves were all high- er than those from roots and stems; the quality of Chenopodium quinoa Willd ge- nomic DNA extracted by modified CTAB method and high-salt Iow-pH method was better, and polyphenols, polysaccharides and other impurities were removed more completely. The PCR-SSR and SSCP detection results showed that the genomic DNA extracted by different methods from different tissues of Chenopodium quinoa Willd all could be better amplified, and high-quality strips could be obtained. So the Chenopodium quinoa Willd genomic DNA extracted by the three methods all can be used for subsequent molecular biology research.

Light-harvesting Polymer Based DNA Assay for SNP Identification of BRCA1 Mutations

We report an improved strategy for conjugated polymer-based optical DNA detection with stem-loop structured DNA probes.The high sensitivity of conjugated polymer-based biosensors arises from light harvesting by the conjugated polymer and the related amplified fluorescent signal transduction either through quenching or through Fǒrster energy transfer) In our previous publication,we demonstrated that the coupling of magnetic particles to conjugated polymers significantly improves the selectivity of this class of DNA sensors.Here we further improved the selectivity by introducing both stem-loop structured capture probe and competition probe.We demonstrated further that this improved magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

Iron-Mediated Oxidative DNA Damage Detected by Fluorometric Analysis of DNA Unwinding in Isolated Rat Liver Nuclei

Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of five antioxidants (catalase, superoxide dismutase, dimethyl sulfoxide, glutathione and diallyl sulfide) on this oxidative nuclear damage were also investigated. At the 0.05 level for statistical significance, iron induced concentration-dependent DNA degradation, and this effect was enhanced by ascorbate and bleomycin. The antioxidants catalase, dimethyl sulfoxide, and diallyl sulfide significantly reduced the iron-ascorbate-induced DNA damage, whereas superoxide dismutase and dimethyl sulfoxide significantly reduced iron-bleomycin-induced damage. Glutathione significantly increased the iron-bleomycin-induced DNA damage. These results suggest that the reactive oxygen species generated by iron, iron-ascorbate, and iron-bleomycin are responsible for the DNA strand breaks in isolated rat liver nuclei.

PCR-based Assay for the Detection of Xanthomonas campestris pv. mangiferaeindicae Causing Bacterial Black Spot in Mango

[Objective] This study aimed to develop a PCR assay for detecting Xanthomonas campestris pv. mangiferaeindicae(Xcm) in culture and in planta. [Method] Primers(Xcm HF and Xcm HR) were designed based on the partial sequence of hrp B gene from xanthomonads to develop a PCR assay for Xcm. Furthermore, specificity and sensitivity of the primer pairs were analyzed in detection of genomic DNA and cell from Xcm. [Result] Amplication was positive only with genomic DNA from positive control ATCC11637 and 12 Xcm strains; no PCR products were amplified with genomic DNA from ten other xanthomonads and seven other bacterial species. The sensitivity of detection was 2.4 pg/μl genomic DNA, and 1.8 × 104CFU/ml cells. The primers also worked well for pathogen detection in direct PCR assays of Xcm colonies grown on liquid medium and in PCR assays of total DNA from leaf, branch and fruit lesions. [Conclusion] A PCR assay was successfully established for rapid detection of Xcm in culture and in planta.

Comparison of benzothiazole-based dyes for sensitive DNA detection

For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most classic DNA stain,but suffers from its high carcinogenicity.A series of less toxic alternatives were developed,many of which contain the core structure of the benzothiazole ring.However,the relationship between the structure and the DNA detection performance was not illustrated.Herein,five benzothiazole dyes,namely thiazole orange,SYBR Green I,Pico Green,SYBR Safe,and thioflavine-T,were compared for DNA detection through direct fluorescence and gel electrophoresis,with particular focus on the structure-performance relationship.It turned out that SYBR Green I is currently the best choice for DNA detection.The results in this work may be useful for future DNA-staining dye developments.

Temperature effect on translocation speed and capture rate of nanopore-based DNA detection

We study the effects of electrolyte temperature on DNA molecule translocation experimentally without and with a temperature gradient across nanopore membranes.The same temperatures on both electrolyte chambers are first considered.The DNA molecule translocation time is measured to be 2.44 ms at 2°C in both chambers,which is 1.57 times longer than at 20°C.Then the temperature difference effect is characterized in both chambers.The results show that the DNA translocation speed can be slowed down as long as one side temperature is lowered,irrespective of the temperature gradient direction.This indicates that the thermophoretic driving force generated by a temperature gradient has no obvious effect on the threading speed of DNA molecules,while the main reason for the slowed DNA translocation speed is the increased viscosity.Interestingly,the capture rate of DNA molecules is enhanced under a temperature gradient condition,and the capture rate during DNA translocation from hot side at 21°C to cold one at 2°C is 1.7 times larger than that under the condition of both chambers at 20°C.Finally,an optimized configuration is proposed to acquire higher capture rates and lower DNA translocation speeds.

Progress in Silicon Nanowire-Based Field-Effect Transistor Biosensors for Label-Free Detection of DNA

Silicon nanowire （SiNW）, as one-dimensional semiconducting nanomaterial, has been incorporated into the filed-effect transistor （FET） devices to increase the efficacy and signal-to-noise in DNA sensing applications. Due to the advantages of high sensitivity, excellent selectivity, label-free detection, direct electrical readout, and minia- turization, SiNW FET-based DNA sensors have been regarded as an important tool in applications of molecular di- agnostics, DNA sequencing, gene expressions, and drug discovery. Here, we review the recent progress in SiNW- FET sensors for label-free electrical DNA detection. We first introduce the working principle of SiNW-FET DNA sensors, SiNW fabrication technologies, bio-functionalization on nanowire surface, and enhancement of device sen- sitivity. Then we sum up the applications of SiNW sensors in detection of DNA hybridization, infectious viruses, microRNA, genetic change （DNA mutation, DNA methylation, and DNA repair）, and protein-DNA interactions. We address several crucial points of sensing performance including sensitivity, selectivity, and limit of detection. Finally, the perspectives, challenges, and some solutions of the field are also discussed.

One-Pot Polymerase Chain Reaction with Gold Nanoparticles for Rapid and Ultrasensitive DNA Detection

We report a novel method for rapid,colorimetric detection of a specific deoxyribonucleic acid(DNA)sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers.Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple.This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary,and is a promising tool for rapid disease diagnostics and gene sequencing.

Plasmonic nanoparticles and nucleic acids hybrids for targeted gene delivery,bioimaging,and molecular recognition

Promising biomedical applications of hybrid materials composed of gold nanoparticles and nucleic acids have attracted strong interest from the nanobiotechnological community.The particular interest is owing to the robust and easy-to-make synthetic approaches,to the versatile optical and catalytic properties of gold nanoparticles combined with the molecular recognition and programmable properties of nucleic acids.The significant progress is made in the develop-ment of DNA-gold nanostructures and their applications,such as molecular recognition,cell and tissue bioimaging,targeted delivery of therapeutic agents,etc.This review is focused on the critical discussion of the recent applications of the gold nanoparticles-nucleic acids hybrids.The effect of particle size,surface,charge and thermal properties on the interactions with functional nucleic acids is discussed.For each of the above topics,the basic principles,recent advances,and current challenges are discussed.Emphasis is placed on the systematization of data over the theranostic systems on the basis of the gold nanoparticles-nucleic acids hybrids.Specifically,we start our discussion with observation of the recent data on interaction of various gold nano-particles with nucleic acids.Further we describe existing gene delivery systems,nucleic acids detection,and bioimaging technologies.Finally,we describe the phenomenon of the polymerase chain reaction improvement by gold nanoparticle additives and its potential underlying mechanisms.Lastly,we provide a short summary of reported data and outline the challenges and perspectives.

Candida accommodates non-culturable Helicobacter pylori in its vacuole-Koch's postulates aren't applicable

The following are the responses to the "letter to the editor"("Helicobacter is preserved in yeast vacuoles! Does Koch's postulates confirm it?") authored by Nader Alipour and Nasrin Gaeini that rejected the methods, results, discussions and conclusions summarized in the review article authored by Siavoshi F and Saniee P. In the article, 7 papers, published between 1998 and 2013, were reviewed. The 7 papers had been reviewed and judged very carefully by the assigned expertise of the journals involved, including the reviewers of the World Journal of Gastroenterology(WJG), before publication. In the review article, 121 references were used to verify the methods, results and discussions of these 7 papers. The review article was edited by the trustworthy British editor of the(WJG), and the final version was rechecked and finally accepted by the reviewers of(WJG). None of the reviewers made comments like those in this "letter to the editor", especially the humorous comments, which seem unprofessional and nonscientific. Above all, the authors' comments show a lack of understanding of basic and advanced microbiology, e.g. bacterial endosymbiosis in eukaryotic cells. Accordingly, their comments all through the letter contain misconceptions. The comments are mostly based on personal conclusions, without any scientific support. It would have been beneficial if the letter had been reviewed by the reviewers of the article by Siavoshi and Saniee.

Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples

In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.

Advances in single-particle detection for DNA sensing

Rapid, accurate and sensitive detection of particular DNA sequence is critical in fundamental biomedical research and clinical diagnostics. However, conventional approaches for DNA assay often suffer from cumbersome procedures, long analysis time and insufficient sensitivity. Recently, single-particle detection technology has emerged as a powerful tool in the biosensing area due to its significant advantages of ultrahigh sensitivity, low sample-consumption and rapid analysis time. Especially, the introduction of novel nanomaterials has greatly promoted the development of single-particle detection and its applications for DNA sensing. In this review, we summarize the recent advance in single-particle detection strategies for DNA sensing, and focus mainly on metallic nanoparticle-and semiconductor quantum dot-based single-particle detection. We highlight the emerging trends in this field as well.

A dual-amplification system for colorimetric DNA detection based on the assembly of biomolecules

A dual-amplification system is reported to apply in DNA sensing via the assembly of DNA and protein. In this process, the biotinylatedcapature DNA bounded with streptavidin（SA） through the biotinstreptavidin reaction, and then the assembly of DNA and protein was triggered by the linker DNA after the target hybridized with biotinylatedcapature DNA. Sequentially, the 3,3＇,5,5＇-tetramethylbenzidine（TMB）was oxidized by H_2O_2 under the catalysis of horseradish peroxidase. Based on the variation of the color and the UV–vis absorbance intensities, qualitative and quantitative DNA analyses were realized. This proposed method could detect the target DNA as low as 1.75 pmol/L and discriminate perfectly matched target DNA from the mismatch DNA. What＇s more, it can be expanded to detect other molecules with a reasonable design of the corresponding DNA sequences.