Gene-Z: a device for point of care genetic testing using a smartphone

By 2012, point of care (POC) testing will constitute roughly one third of the $59 billion in vitro diagnostics market. The ability to carry out multiplexed genetic testing and wireless connectivity are emerging as key attributes of future POC devices. In this study, an inexpensive, user-friendly and compact device (termed Gene-Z) is presented for rapid quantitative detection of multiple genetic markers with high sensitivity and specificity. Using a disposable valve-less polymer microfluidic chip containing four arrays of 15 reaction wells each with dehydrated primers for isothermal amplification, the Gene-Z enables simultaneous analysis of four samples, each for multiple genetic markers in parallel, requiring only a single pipetting step per sample for dispensing. To drastically reduce the cost and size of the real-time detector necessary for quantification, loop-mediated isothermal amplification (LAMP) was performed with a high concentration of SYTO-81, a non-inhibiting fluorescent DNA binding dye. The Gene-Z is operated using an iPod Touch, which also receives data and carries out automated analysis and reporting via a WiFi interface. This study presents data pertaining to performance of the device including sensitivity and reproducibility using genomic DNA from Escherichia coli and Staphylococcus aureus. Overall, the Gene-Z represents a significant step toward truly inexpensive and compact tools for POC genetic testing.     

An integrated microfluidic device for the simultaneous detection of multiple antibiotics

Residual antibiotics in food pose a serious long-term threat to human health. Therefore, an on-site visualization method for antibiotic detection is required. However, the requirements of traditional antibiotic testing methods in terms of operator proficiency and equipment cost hinder the rapid point-of-care-testing detection of suspected samples. Herein, we reported an integrated microfluidic device combining a microfluidic chip containing cruciform valves with immunochromatographic strips for the rapid detection of multiple antibiotics in milk. The rapid qualitative and quantitative analysis of four types of antibiotics (sulfonamides, β-lactams, streptomycin, and tetracyclines) was performed using mobile phone photography and mobile phone application analysis. The detection time was maintained at 10 min. The limits of detection (LODs) for the four antibiotics were 0.15, 0.12, 0.25, and 0.29 ng/mL, respectively, and the selectivity for the different antibiotics was observed even in a highly complex matrix. This device successfully integrated separation and real-time detection onto a chip and might provide a promising perspective for the detection of multiple antibiotics in milk.     

Cardiac markers: a clear cause for point-of-care testing

Point-of-care testing (POCT) in patients with ischemic heart disease is driven by the time-critical need for fast, specific, and accurate results to initiate therapy instantly. According to current guidelines, the results of the cardiac marker testing should be available to the physician within 30 min (“vein-to-brain” time) to initiate therapy within 60–90 min (“door-to-needle” time) after the patient has arrived at the emergency room or intensive care unit. This article reviews the current efforts to meet this goal (1) by implementing POCT of established biochemical markers such as cardiac troponins, creatine kinase MB, and myoglobin, in accelerated diagnosis and management workflow schemes, (2) by improving current POCT methods to obtain more accurate, more specific, and even faster tests through the integration of optical and electrochemical sensor technology, and (3) by identifying new markers for the very early and sensitive detection of myocardial ischemia and necrosis. Furthermore, the specific requirements for cardiac POCT in regard to analytical performance, comparability, and diagnostic sensitivity/specificity are discussed. For the future, the integration of new immunooptical and electrochemical chip technology might speed up diagnosis even further. However, every new development will have to meet the stringent method validation criteria set for corresponding central laboratory testing.     

基于双纳米金探针杂交法检测HBV DNA

利用双纳米金探针结合基因芯片平台建立了一种检测乙肝病毒基因(HBV DNA)的新方法. 根据HBV DNA的保守序列设计捕获探针和信号报告探针, 通过一对互补的纳米金检测探针的双杂交法对HBV DNA进行信号放大, 最后进行银染, 达到对HBV DNA的可视化检测. 该方法的灵敏度高, 可检测10 fmol/L的HBV DNA, 且能在1.5 h内完成检测. 其具有的快速、 高灵敏度及低成本等优势使其有望发展成为一种检测HBV DNA的新方法.     

Functional integration of DNA purification and concentration into a real time micro-PCR chip

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.     

Single-molecule immunoassay and DNA diagnosis

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.     

发铅检测法的临床应用价值评估

从铅中毒发铅诊断标准、发铅诊断试验及发铅诊断临床应用3个方面论述了发铅检测法在铅中毒诊断、筛查和监督中的实际应用问题。根据临床经验和发铅-血铅比值确定了居民发铅正常值上限及铅中毒发铅诊断分级标准;即使以血铅测定值为"金标准",诊断试验表明,发铅测定在铅中毒诊断中仍有实际应用价值;发铅检测法自上世纪60年代以来一直沿用至今,绝大多数研究者认为,发铅测定是诊断、筛查铅中毒和监督环境铅污染最简单、有效的工具。     

Biosensors and rapid diagnostic tests on the frontier between analytical and clinical chemistry for biomolecular diagnosis of dengue disease: a review

The past decades have witnessed enormous technological improvements towards the development of simple, cost-effective and accurate rapid diagnostic tests for detection and identification of infectious pathogens. Among them is dengue virus, the etiologic agent of the mosquito-borne dengue disease, one of the most important emerging infectious pathologies of nowadays. Dengue fever may cause potentially deadly hemorrhagic symptoms and is endemic in the tropical and sub-tropical world, being also a serious threat to temperate countries in the developed world. Effective diagnostics for dengue should be able to discriminate among the four antigenically related dengue serotypes and fulfill the requirements for successful decentralized (point-of-care) testing in the harsh environmental conditions found in most tropical regions. The accurate identification of circulating serotypes is crucial for the successful implementation of vector control programs based on reliable epidemiological predictions. This paper briefly summarizes the limitations of the main conventional techniques for biomolecular diagnosis of dengue disease and critically reviews some of the most relevant biosensors and rapid diagnostic tests developed, implemented and reported so far for point-of-care testing of dengue infections. The invaluable contributions of microfluidics and nanotechnology encompass the whole paper, while evaluation concerns of rapid diagnostic tests and foreseen technological improvements in this field are also overviewed for the diagnosis of dengue and other infectious and tropical diseases as well.     

基于微流控芯片的72重单核苷酸多态性族群推断系统的构建

建立了常染色体单核苷酸多态性（SNPs）复合检测芯片体系,用于未知个体的族群来源推断。基于前期筛选的74-SNPs组合,采用竞争性等位基因特异性聚合酶链式反应（PCR）的原理构建SNPs的扩增体系,在微流控芯片的每个反应孔内完成一个SNP的检测,通过高通量PCR微流控芯片实现了其中72个SNPs的同步检测。芯片的扩增由平板PCR仪完成,反应孔的荧光信号通过激光共聚焦扫描仪检测,最终通过提取的荧光值进行结果分析。使用该芯片检测获得52份样本的SNPs分型,分型结果的准确率为100%。以57个人群的3628个样本为参考人群数据库,进行20份样本的族群来源推断,推断结果与样本的实际来源一致。本研究建立的常染色体72个SNPs微流控芯片体系可以有效地进行SNP多态性分析检测,基于参考数据库,20份检测样本族群推断的准确性为100%。     

A robust sensor platform for label-free detection of anti-Salmonella antibodies using undiluted animal sera

We present a portable and easy-to-use biosensor platform, allowing for label-free detection of diagnostic markers in undiluted animal serum. Exemplarily, this is shown for the detection of anti-Salmonella antibodies. 1-lambda-Reflectometry was used as detection method, making the new biosensor platform portable, cheap, and robust. As recognition elements, lipopolysaccharides (LPSs) from Salmonella typhimurium bacteria were immobilized as sensitive layer on the transducer to carry out serological tests via a direct assay format. For this purpose, a new surface preparation protocol has been worked out allowing for immobilization of the LPS via hydrophobic interactions. It has been shown that results obtained by 1-lambda-Reflectometry are equivalent to those obtained by the non-portable Reflectometric Interference Spectroscopy setup. The new sensor platform was calibrated in both matrices, buffer and undiluted serum. Good sensitivity, selectivity and intra chip reproducibility have been observed. Furthermore, inter chip reproducibility was examined and recovery rates were found to be between 99 and 117 % in undiluted serum.     

A novel colorimetric PCR-based biosensor for detection and quantification of hepatitis B virus

Hepatitis B virus (HBV) can cause viral infection that attacks the liver and it is a major global health problem that put people at a high risk of death from cirrhosis of the liver and liver cancer. HBV has infected one third of the worldwide population, and 350 million people suffer from chronic HBV infection. For these reasons, development of an accurate, sensitive and expedient detection method for diagnosing, monitoring and assessing therapeutic response of HBV is very necessary and urgent for public health and disease control. Here we report a new strategy for detection of viral load quantitation of HBV based on colorimetric polymerase chain reaction (PCR) with DNAzyme-containing probe. The special DNAzyme adopting a G-quadruplex structure exhibited peroxidase-like activity in the presence of hemin to report colorimetric signal. This method has shown a broad range of linearity and high sensitivity. This study builds important foundation to achieve the specific and accurate detection level of HBV DNA with a low-cost and effective method in helping diagnosing, preventing and protecting human health form HBV generally all over the world and especially in developing countries.     

Comparative proteomic analysis of drug sodium iron chlorophyllin addition to Hep 3B cell line

The human hepatoma 3B cell line was chosen as an experimental model for in vitro test of drug screening. The drugs included chlorophyllin and its derivatives such as fluo-chlorophyllin, sodium copper chlorophyllin, and sodium iron chlorophyllin. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method was used in this study to obtain the primary screening results. The results showed that sodium iron chlorophyllin had the best LC(50) value. Proteomic analysis was then performed for further investigation of the effect of sodium iron chlorophyllin addition to the Hep 3B cell line. The proteins identified from a total protein extract of Hep 3B before and after the drug addition were compared by two-dimensional-gel-electrophoresis. Then 32 three-fold differentially expressed proteins were successfully identified by MALDI-TOF-TOF-MS. There are 29 unique proteins among those identified proteins. These proteins include proliferating cell nuclear antigen (PCNA), T-complex protein, heterogeneous nuclear protein, nucleophosmin, heat shock protein A5 (HspA5) and peroxiredoxin. HspA5 is one of the proteins which are involved in protecting cancer cells against stress-induced apoptosis in cultured cells, protecting them against apoptosis through various mechanisms. Peroxiredoxin has anti-oxidant function and is related to cell proliferation, and signal transduction. It can protect the oxidation of other proteins. Peroxiredoxin has a close relationship with cancer and can eventually become a disease biomarker. This might help to develop a novel treatment method for carcinoma cancer.     

高灵敏纳米金比色芯片检测乙型肝炎病毒DNA及其变异

构建了新型纳米金比色芯片,利用Taq DNA连接酶的连接特异性,将其与乙型肝炎病毒DNA( HBV-DNA)靶序列完全互补杂交的捕获探针(固定在芯片上)和纳米金修饰的探针连接成一条链,从而将纳米金颗粒固定到芯片点阵上,再通过银染反应放大,形成裸眼可见的显色信息.通过点阵的位置及灰度,即可判断HBV-DNA靶序列的单碱基突变,并得出相对定量信息.本实验对不同浓度的HBV-DNA靶序列进行了检测.结果显示:此技术对单碱基突变有很强的特异性识别能力,并且具有较高的灵敏度(约10 pmol/L),在10～100 pmol/L浓度范围内表现出较好的线性关系.该技术检测时间短(＜1 h)、操作简单、不需要特殊的检测设备,具有很好的临床应用前景.     

A bio-barcode assay for on-chip attomolar-sensitivity protein detection

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.     

A novel cartridge for nucleic acid extraction,amplification and detection of infectious disease pathogens with the help of magnetic nanoparticles

Nucleic acid detection (NAD) based on real-time polymerase chain reaction (real-time PCR) is gold standard for infectious disease detection. Magnetic nanoparticles (MNPs) are widely used for nucleic acid extraction (NAE) because of their excellent properties. Microfluidic technology makes automated NAD possible. However, most of the NAD microfluidic chips are too complex to be applied to point-of-care (POC) testing. In this paper, a simple-structure cartridge was developed for POC detection of infectious diseases. This self-contained cartridge can be divided into a magnetic-controlled NAE part, a valve-piston combined fluidic control part and a PCR chip, which is able to extract nucleic acid from up to 500 µL of liquid samples by MNPs and finish the detection process from “sample in” to “answer out” automatically. Performance tests of the cartridges show that it met the demands of automated NAD. Results of on-cartridge detection of hepatitis B virus (HBV) demonstrated that this system has good uniformity and no cross-contamination between different cartridges, and the limit of detection (LOD) of this system for HBV in serum is 50 IU/mL. Multiplex detections of severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) with a concentration of 500 copies/mL were carried out on the system and 100% positive detection rate was achieved.     

A simple and sensitive solid-phase radioimmunoassay for the assay of human TSH antibody

A simple and sensitive radioimmunoassay procedure is described for the screening and detection of specific antibodies in hybridoma cell lines. The specific procedure was developed to screen for antibodies against human thyrotropin (hTSH), but the procedure is applicable to screening for any desired antibodies. The immunoglobulin G(IgG) fraction of goat anti-mouse IgG is used to coat wells of microtiter plates. Anti-hTSH antibodies are measured by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated hTSH. Unlabeled hTSH may also be detected by its ability to inhibit binding of 125I-hTSH to the coated wells. This assay technique meets the demands of simplicity, sensitivity, reproducibility, and rapidity as a screening assay of hybridoma cell lines capable of secreting anti h-TSH.     

A novel high-resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA

There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.     

Rapid and sensitive handheld biosensor for detection of hantavirus antibodies in wild mouse blood samples under field conditions

Hantavirus cardiopulmonary syndrome (HCPS) is an acute, life threatening viral illness that was first recognized in the spring of 1993 during an outbreak in the Four Corners region of the southwest United States. The etiologic agent, Sin Nombre (SN) virus, is a zoonotic infection that is enzootic in deer mice (Peromyscus maniculatus). Both human and rodent infections lead to specific antibody responses, and detection of such antibodies is the mainstay of diagnosis of infection in both species. More than 300 cases of HCPS have been confirmed in 30 states and four Canadian provinces since 1993 outbreak. Forty percent have been fatal. Conventional methods for the detection of hantavirus antibodies require 4-9 h. Since HCPS can rapidly progress to death, there is a clear need for a rapid diagnostic test. Rapid diagnostic tests for the detection of hantavirus infection should be ideally be (a) quick (b) simple to carry out, (c) allowing if possible for use in field conditions. A field-deployable test would have a particular advantage for diagnosis of infection in wild rodents, such as for risk assessments around case-households, as well as potential human diagnostic applications in battlefield situations. A prototype amperometric immunosensor based on highly dispersed immunoelectrode is developed for operation in field conditions on the importance of rapid and sensitive assay for hantavirus infection in mouse blood serum. This study was initiated to simplify and develop effective procedures for screening rodent serum samples in remote areas. A sandwich scheme immunoassay has been developed and used. Naphthol formed as a result of enzymatic reduction of naphthyl phosphate in the presence of alkaline phosphatase label has been detected amperometrically. The performance of the biosensor device is evaluated by testing mice blood samples in the wild under field conditions. The results were compared with the results obtained using the strip immunoblot assay (SIA) and that obtained from Centers for Disease Control and Prevention (CDC). The overall time of analysis is as low as 25 min and statistical analysis of the data indicated promising results. The device can be easily adapted for fast analysis of other microorganisms under non-laboratory conditions, in remote locations and field operations.     

New fluorogenic probes for oxygen and carbene transfer: a sensitive assay for single bead-supported catalysts.

A high-throughput screening assay for atom transfer catalysis has been developed. This assay is based on two probes, developed herein, which generate highly fluorescent products upon carbene or oxygen atom transfer. The emission wavelength of probes 1 and 5 shift significantly (up to 90 nm) upon epoxidation, allowing detection of product at 3% conversion. Probe 7 is not fluorescent, while fluorescence emission by carbene insertion/rearrangement product 8 allows detection at less than 1% conversion. Such sensitivity allows for examination of single-bead reactions in a high throughput array format (1536 wells per plate), and provides a broad detection window ranging from single to high turnover numbers. Thousands of metal complexes are evaluated in a single screening experiment. Preliminary screening of a diverse ligand library with probe 7 in the presence of Rh(II) uncovered new catalysts capable of cyclopropanation and C-H insertion.