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.

Robust and efficient direct multiplex amplification method for large-scale DNA detection of blood samples on FTA cards

Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.

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.

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.

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。

血清CYFRA21-1、CA125联合HPV DNA检测在宫颈癌早期筛查中的价值及病理特征研究

目的分析血清细胞角蛋白19片段(CYFRA21-1)、糖类抗原125(CA125)联合HPV DNA检测在宫颈癌早期筛查中的价值及病理特征。方法选取2019年8月至2023年6月沧州市中心医院收治的宫颈癌患者152例为观察组,另选取同期在本院行体检且各项正常女性80名为对照组;对比两组血清CYFRA21-1、CA125水平以及HPV DNA检测结果;对比观察组不同手术病理结果及血清CYFRA21-1、CA125表达水平以及HPV DNA检测结果;以病理学检查为金标准,分析血清CYFRA21-1、CA125水平以及HPV DNA检测单独以及联合诊断宫颈癌的一致性;绘制ROC曲线,分析血清CYFRA21-1、CA125联合HPV DNA单独检测及联合检测宫颈癌的效能。结果152例患者中,鳞状细胞癌104例,腺癌患者48例;其中轻度不典型增生66例、中度不典型增生57例、重度不典型增生29例。观察组血清CYFRA21-1、CA125水平以及HPV DNA阳性率均高于对照组,差异有统计学意义(P<0.05);血清CYFRA21-1、CA125水平:鳞状细胞癌<腺癌,轻度不典型增生<中度不典型增生<重度不典型增生,差异均有统计学意义(P<0.05)。观察组不同分期、不同肿瘤增生类型的HPV DNA阳性率比较,差异无统计学意义(P>0.05);血清CYFRA21-1、CA125水平、HPV DNA检测单独以及联合诊断宫颈癌与病理学检查结果的一致性Kappa值分别为0.677、0.731、0.756、0.902;CA125+CYFRA21-1+HPV DNA联合检测的AUC为0.894,高于CA125、CYFRA21-1、HPV DNA单独检测(P>0.05)。结论血清CYFRA21-1、CA125联合HPV DNA检测的诊断效能高于单一检测,提示三指标联合检测可显著提高宫颈癌早期筛查的诊断价值,可为制定临床治疗方案提供参考资料。

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.

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.

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.

不同核酸提取方法对HBV-DNA检测性能验证情况分析

目的评估2种乙型肝炎病毒(HBV)-DNA提取方法及2家检测试剂的性能,有助于选择优化提取试剂和检测试剂。方法2023年4月采用达安全自动核酸提取仪提取法(磁珠法)和手工提取法(一步法),并用达安和圣湘2种HBV-DNA试剂检测,对其进行精密度、正确度、线性范围、检出限及抗干扰能力等性能进行验证和评价。结果达安全自动核酸提取仪提取达安试剂检测、手工提取达安试剂检测和手工提取圣湘试剂检测在精密度、正确度、线性范围、检出限方面验证结果均达标;达安全自动核酸提取仪提取圣湘试剂检测在低值检测中变异系数大于5%,最低检测限验证不合格;抗干扰能力方面,全自动核酸提取仪提取的2.0 g/dL血红蛋白浓度的样本用达安和圣湘试剂检测结果均不受影响。手工提取甘油三酯浓度达3000 mg/dL的样本用达安和圣湘试剂检测的结果均不受影响。结论不同厂家的提取和检测试剂避免混用,达安和圣湘试剂对HBV-DNA定量检测的结果均符合要求。

Surface modification of nanogold particles in DNA detection with quartz crystal microbalance

Nanogold particles in different sizes, from 5 to 60 nm, were utilized to modify the surface of the quartz crystal microbalance (QCM). It was found that the gold-particle size of the modified QCM affects both the amount of the immobilization of the probe on the surface of QCM and the hybridization rate of the target DNA. 20 nm was determined to be the optimal size for the surface modification and it can maximally increase the sensitivity of the DNA detection.

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.

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.

氧化石墨烯-DNA纳米探针用于三磷酸腺苷的检测与药物递送

癌细胞内三磷酸腺苷(ATP)浓度异常与肿瘤发生发展过程密切相关,因此,快速、准确地检测细胞内外ATP水平具有重要意义。盐酸阿霉素(DOX)是一种广泛使用的抗癌药物,能嵌入DNA碱基对,并通过抑制DNA复制和转录诱导细胞凋亡。氧化石墨烯(Graphene oxide, GO)由于具有毒性低、比表面积大和易功能化,可以有效、稳定地负载DNA纳米探针进入细胞等优点而被广泛应用。然而,复杂环境中的生物分子容易通过物理吸附竞争性结合到GO表面,导致假阳性信号。基于此,提出了一种新型的GO-DNA纳米探针,并将其应用于ATP的检测与抗癌药物DOX靶向递送。以ATP的核酸适配体与其互补链杂交形成双链DNA(dsDNA),并通过G-C碱基对负载DOX,利用互补链延伸的poly A序列吸附到GO表面构建了GO-dsDNA-DOX纳米探针,能极大程度地降低复杂环境中物理吸附引起的干扰,减少假阳性信号产生。ATP与适配体特异性结合会导致DOX释放,根据其荧光“off-on”实现ATP的定量分析,DOX荧光强度与ATP含量在0.08~8.0 mmol/L范围内呈现良好的线性关系,线性方程为IF=3.0897c+129.08,检测限(3σ/k)为0.059 mmol/L,且方法具有良好的选择性和抗干扰能力。细胞毒性及荧光成像结果表明,负载DOX的纳米探针在人乳腺癌细胞(MCF-7)内有明显的药物释放,显著诱导细胞凋亡。该研究建立了一种免修饰、简单和快速的ATP含量检测分析方法,并利用癌细胞内高浓度ATP实现靶向药物递送,降低了对正常细胞的毒副作用,为癌症的治疗提供了新思路。

DNA methylation detection methods used in colorectal cancer

Colorectal cancer(CRC)remains a major contributor to the number of cancerrelated deaths that occur annually worldwide.With the development of molecular biology methods,an increasing number of molecular biomarkers have been identified and investigated.CRC is believed to result from an accumulation of epigenetic changes,and detecting aberrant DNA methylation patterns is useful for both the early diagnosis and prognosis of CRC.Numerous studies are focusing on the development of DNA methylation detection methods or DNA methylation panels.Thus,this review will discuss the commonly used techniques and technologies to evaluate DNA methylation,their merits and deficiencies as well as the prospects for new methods.

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.

循环肿瘤DNA的检测技术及在癌症诊疗中的应用价值

循环肿瘤DNA(ctDNA)来源于肿瘤,能很好地反映肿瘤的基因信息,并且会随着肿瘤的进展发生相应的改变。近年来,ctDNA独特的能力备受人们关注并被广泛研究,本文在总结ctDNA的来源、性质和样品处理的基础上,对ctDNA的检测技术及在癌症诊疗中的应用进行综述,阐述了ctDNA标准物质在二代测序中的作用及重要性,并指出ctDNA样品的收集、存储、定量检测以及数据分析等各个流程中建立统一标准及规范的迫切性。

产前胎儿染色体异常诊断中无创DNA检测与羊水穿刺的应用效果比较

目的:比较产前胎儿染色体异常诊断中无创DNA检测与羊水穿刺的应用效果。方法:回顾性分析因无创高危就诊优生遗传科行羊水穿刺的512例孕妇病历资料,所有患者均接受无创DNA产前检测和羊水穿刺进行核型和染色体微阵列分析(CMA)。探究无创DNA产前检测染色体异常符合率。结果:①无创DNA产前检测提示:21三体高危138例(26.95%),18三体高危54例(10.55%),13三体高危27例(5.27%),性染色体高危167例(32.62%),其他常染色体高危126例(24.61%);②羊水穿刺核型诊断:染色体异常210例,总诊断符合率41.02%(210/512),包括21三体综合征胎儿105例,诊断符合率76.09%(105/138),确诊18三体综合征胎儿14例,诊断符合率25.93%(14/54),确诊13三体综合征胎儿3例,诊断符合率11.11%(3/27),确诊性染色体异常胎儿62例,诊断符合率37.13%(62/167),确诊其他常染色体异常胎儿26例,诊断符合率20.63%(26/126),不同染色体高危风险分布及诊断符合率差异有统计学意义(Z=5.391,P=0.000;χ^(2)=117.184,P=0.001);③随访结局:确诊21三体综合征胎儿孕妇引产102例,生产3例;确诊18三体综合征胎儿孕妇引产13例,术后流产1例;确诊13三体综合征胎儿孕妇均引产;确诊性染色体异常胎儿孕妇引产36例,生产26例;确诊其他常染色体异常胎儿孕妇引产10例,生产26例。结论:产前胎儿染色体异常诊断中使用无创DNA检测可有效筛出21三体综合征胎儿,但对其他染色体异常检出率较低,临床产前检查仍需要联合羊水穿刺诊断。

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.

A simple visual method for DNA detection based on the formation of gold nanoparticles

A simple visual method for DNA detection during the formation of gold nanoparticles(AuNPs) was developed based on different electrostatic properties of single strand DNA(ssDNA) and double strand DNA(dsDNA).Since the ssDNA is easy to bind to AuNPs due to its exposed bases which could prevent saltinduced aggregation of AuNPs.The dsDNA always present negative charge because its negatively charged phosphate backbone is exposed.In this case,the dsDNA could disturb the adsorption between dsDNA and AuNPs and result in non-aggregation of AuNPs.After hybridization,chloroauric acid and ascorbic acid were added to the mixture solution,and the solution changed to red immediately and turned to purple in10 min in the present of target DNA.TEM results confirmed that the change of color stemed from aggregation of AuNPs.In order to obtain accurate results by naked eye,the DNA detection assay should be conducted under pH 7.0.