Low noise bipolar photodiode array protein chip based on on-chip bioassay for the detection of <Emphasis Type="Italic">E. coli</Emphasis> O157:H7

A bipolar photodiode array (PDA) protein chip is presented for the detection of E. coli O157:H7. Through unique design of the bipolar PDA microchip, the device was able to detect E. coli O157:H7 directly on the surface of the bipolar PDA. The bipolar PDA microchip maintained low noise level in the entire process of on-chip protein assay and demonstrated high performance of analog signal processing. At every reaction step of the on-chip bioassay, stability of wet photodiode detection elements was confirmed by monitoring the variance of their photosignals with respect to the irradiated red beam. The background signal represented less than 1.8% variance with respect to maximum signal of photodiode detection elements. As a result of using the on-chip bioassay, any complicated optical alignment and components could be removed in the constructed protein chip. This protein chip enables direct optical detection of E. coli O157:H7 eliminating the need of conventional expensive microplate reader that is incompatible with size of sampling platform of protein chip. The independence of the constructed protein chip on conventional microplate reader can contribute greatly to further miniaturization of protein chip and field usable lab-on-a-chip.     

A flexible implantable microelectrode array for recording electrocorticography signals from rodents

Biomedical Microdevices - Electrocorticography signals, the intracranial recording of electrical signatures of the brain, are recorded by non-penetrating planar electrode arrays placed on the...     

Self-calibrating interference refractometer using a linear photodiode array for anaesthetic gas analysis

The laboratory standard for measurement of gas concentrations in binary mixtures is the manually operated interfence refractometer. We describe an automatic interference refractometer for theatre use incorporating a linear photodiode array and using digital electronics for signal analysis. The support system performs automatic self-calibration, samples gases from an anaesthetic machine, feeds them to the refractometer and presents information on gas concentrations and possible alarm conditions to the anaesthetist. The instrument may be incorporated into a new anaesthetic machine or may be an addition to an existing one. The instrument may also be applied in other fields where concentrations of known gases need to be monitored automatically.     

AFP电化学发光免疫分析方法的建立

目的建立一种快速、特异、灵敏的检测人甲胎蛋白（AFP）的电化学发光免疫分析法。方法用链霉亲和素包被的磁性微粒、生物素标记的抗AFP单克隆抗体、钌复合物标记配对的抗AFP单克隆抗体组成AFP电化学发光免疫分析法试剂,在电化学发光免疫分析仪上对其准确性、灵敏度、特异性等效能进行方法学评价,同时用所建方法与进口的同类电化学发光免疫分析法试剂（Roche）对80例肝癌患者血清AFP检测结果进行相关性分析。对218名健康志愿者血清AFP进行了正常值调查。结果自建电化学发光免疫分析法检测AFP的批内精密度在2.8%～4.5%之间,批间精密度在3.2%～9.8%之间,分析灵敏度为0.605ng/ml。与进口同类试剂比较,直线回归方程为y=0.9936x-0.4566（r=0.9877,P〈0.05）。分析测量范围为0.605～1452.0ng/ml,自建试剂与CEA和CA199无交叉反应。自建电化学发光免疫分析法检测AFP的正常参考值为〈6.7ng/ml。结论自建AFP电化学发光免疫分析法特异性高,灵敏度好,与进口同类试剂检测结果相关性达0.9877。确定的实验室正常参考范围接近进口同类试剂,具备产业化的潜能。     

Tape underlayment rotary-node (TURN) valves for simple on-chip microfluidic flow control

We describe a simple and reliable fabrication method for producing multiple, manually activated microfluidic control valves in polydimethylsiloxane (PDMS) devices. These screwdriver-actuated valves reside directly on the microfluidic chip and can provide both simple on/off operation as well as graded control of fluid flow. The fabrication procedure can be easily implemented in any soft lithography lab and requires only two specialized tools—a hot-glue gun and a machined brass mold. To facilitate use in multi-valve fluidic systems, the mold is designed to produce a linear tape that contains a series of plastic rotary nodes with small stainless steel machine screws that form individual valves which can be easily separated for applications when only single valves are required. The tape and its valves are placed on the surface of a partially cured thin PDMS microchannel device while the PDMS is still on the soft-lithographic master, with the master providing alignment marks for the tape. The tape is permanently affixed to the microchannel device by pouring an over-layer of PDMS, to form a full-thickness device with the tape as an enclosed underlayment. The advantages of these Tape Underlayment Rotary-Node (TURN) valves include parallel fabrication of multiple valves, low risk of damaging a microfluidic device during valve installation, high torque, elimination of stripped threads, the capabilities of TURN hydraulic actuators, and facile customization of TURN molds. We have utilized these valves to control microfluidic flow, to control the onset of molecular diffusion, and to manipulate channel connectivity. Practical applications of TURN valves include control of loading and chemokine release in chemotaxis assay devices, flow in microfluidic bioreactors, and channel connectivity in microfluidic devices intended to study competition and predator/prey relationships among microbes.     

Comparison of an electrochemiluminescence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain (RCA-B)

An electrochemiluminescence (ECL) assay for the detection of the B chain of ricin (RCA-B) in a 96-well plate format was developed in parallel with a colorimetric ELISA utilizing the same pair of antibodies. Sensitivity results were interpreted with the ANOVA and Tukey statistical tests, allowing a direct comparison between the two technologies, that can probably be extended to other protein antigens such as toxins. Reproducibility, repeatability and rapidity of the two techniques were also compared. The ELISA assay utilized an alkaline phosphatase conjugate for signal generation. After optimization, its limit of detection was 400 pg of RCA-B per ml buffer, with an intra-day standard deviation (SD) of 2.2% of the mean and an inter-day SD of 5.1%. The ECL assay utilized ruthenylated antibodies for detection. The ECL measurement was carried out using a Sector PR 400 plate reader. After optimization, its limit of detection was 50 pg of RCA-B per ml buffer, with an intra-day SD of 4.1% of the mean and an inter-day SD of 4.3%. Starting from a pre-coated plate, the ELISA assay was completed in 7 h and the ECL assay took 2.5 h. While reproducibility and repeatability of the two assays were equivalent, this ECL assay in plate format had an 8-fold better sensitivity for RCA-B detection than the colorimetric ELISA in buffer and in various matrices. The ECL assay was also three times faster, and retained the robustness and convenience of the 96-well plate format.     

Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection

Positional and geometric isomers of mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms were separated on commercially available reversed-phase columns in gradient systems composed of acetonitrile and water, utilizing photodiode array detection. The biological samples were hydrolyzed with 2 M NaOH for 35-40 min at 85-90 degrees C. After cooling, the hydrolysates were acidified with 4 M HCl and the free fatty acids were extracted with dichloromethane. The organic solvent was removed in a gentle stream of argon. The fatty acids were determined after pre-column derivatization with dibromacetophenone in the presence of triethylamine. The reaction components were mixed and reacted for 2 h at 50 degrees C. Separations of derivatized fatty acids were performed on two C18 columns (Nova Pak C18, 4 microm, 250x4.6 mm, Waters) by binary or ternate gradient programs and UV detection at 254 and 235 nm. The geometric and positional isomers of some unsaturated fatty acids were substantially retained on the C18 columns and were distinct from some saturated fatty acids, endogenous substances in biological samples or background interference. Only slight separation of critical pairs of cis-9 C18:1/cis-11 C18:1 and cis-6 C18:1/trans-11 C18:1 was obtained. A ternate gradient program can be used for complete fractionation of a mixture of conjugated linoleic acid isomers (CLA) from cis-9, cis-12 and trans-9, trans-12 isomers of C18:2. The CLA isomers in the effluent were monitored at 235 nm. The CLA isomers were differentiated from saturated and unsaturated fatty acids using a photodiode array detector. The utility of the method was demonstrated by evaluating the fatty acid composition of duodenal digesta, rapeseed and maize oils.     

量子点编码微球蛋白芯片检测丙型肝炎病毒方法的建立

目的 建立检测丙型肝炎病毒(HCV)的量子点编码微球蛋白芯片方法.方法 利用量子点编码微球芯片和免疫荧光技术将高度纯化的HCV NS3、NS4、NS5和Core共价偶联到4种不同颜色编码微球表面,用免疫荧光法检测抗HCV抗体;以建立的量子点编码微球蛋白芯片方法对120例重组免疫印迹法(recombinant immunoblot assay,RIBA)确认的HCV阳性患者和50例阴性患者进行检测,计算所建立的方法的检测敏感度、特异度和准确性.结果 量子点编码微球蛋白芯片检测的敏感度为97.50％ (117/120),特异度为96.0％ (48/50),准确性为97.06％[(117 +48)/(120 +50)];微球蛋白芯片的检测结果与RIBA法相当;分片段抗体检出率HCV Core＞ NS3＞ NS4＞ NS5,检出率分别为92.50％ (111/120)、89.17％ (107/120)、70.83％ (85/120)、52.50％ (63/120).结论 成功地建立了量子点编码微球蛋白芯片方法,操作简便、多元检测、速度快,具有较高的敏感度和特异度以及可重复性.     

A hybrid poly(dimethylsiloxane) microsystem for on-chip whole blood filtration optimized for steroid screening

Miniaturized biochemical devices in glass, silicon and polymer materials are starting to find their way from the academic laboratories to real-life applications. However, most attention has been given to miniaturize the downstream functions of various microfluidic systems, leaving the sample introduction and preparation steps to more conventional, bulkier solutions. For point-of-care diagnostics in particular, it becomes crucial to be able to handle complex human samples in a miniaturized format. In this work, we report on a microsystem for on-chip sample preparation that is able to remove blood cells from whole blood. The hybrid system consists of a commercially available membrane filter incorporated into a poly(dimethylsiloxane) (PDMS) casted device. Membrane materials were evaluated on the bases of low nonspecific adsorption of free and protein-bound testosterone as analyte substance. The hybrid system including a hydrophilic polypropylene filter successfully removed blood cells from diluted human whole blood. Surface oxidation was sufficient to make the plasma filtrate flow through the membrane filter and the channel system by capillary force alone and thus no external pumping source was needed.     

Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin

We compared the ELISA and electrochemiluminescence (ECL) immunoassay technologies for the detection of botulinum type B neurotoxin (BotNT B), which requires highly sensitive techniques due to its potent biological activity. BotNT B complexes are the naturally secreted form of the toxin, approximately a third of which consists of the neurotoxin itself; they were aliquoted and frozen for this study. Results of both techniques were interpreted with the same standard statistical tests (ANOVA and Tukey). We first compared two commercial assays for BotNT B: the detection limit of the colorimetric ELISA was 1.56 ng/ml BotNT B complexes versus 0.39-0.78 ng/ml in the ECL test. We then used the same monoclonal antibody and the same polyclonal antibody, respectively purified by protein A and protein G chromatography, to optimize an in-house ELISA test and an in-house ECL test, making it possible to directly compare the two technologies without interference due to the properties of the antibodies used in the two tests. The colorimetric in-house ELISA had a detection threshold of 3.12 ng/ml versus the in-house ECL test whose detection threshold was 0.78-1.56 ng/ml. Thus, in both cases, the ECL assay was two to four times more sensitive than the colorimetric ELISA. The ECL assay was also more rapid (2.5 h for the in-house ECL versus 5 h for in-house ELISA with precoated wells). Overall, these elements can be used to compare the qualities of the two technologies, at least for the detection of protein antigens such as toxins.     

SRS MapCHECK半导体矩阵用于射波刀脊柱计划剂量验证的应用研究

目的:评估SRS MapCHECK半导体矩阵探测器用于射波刀脊柱计划剂量验证工作的适用性。方法:将SRS MapCHECK探测器安装于专用模体StereoPHAN开展研究工作,测试了射波刀固定和Iris可变准直器的单野计划,以及脊柱临床计划的质量保证（QA）计划,采用SNC Patient软件对比分析实测与计划剂量分布之间的差异,分别计算在2 mm/5%、2 mm/3%和2 mm/2%标准下的γ通过率。结果:在绝对剂量分析模式2 mm/5%,2 mm/3%和2 mm/2%标准下,固定和Iris可变准直器单野计划的γ通过率均为100.0%,脊柱QA计划的平均γ通过率分别为（99.3±1.2）%、（96.5±2.7）%和（93.5±5.3）%。结论:SRS MapCHECK半导体矩阵探测器在2 mm/5%和2 mm/3%的γ分析标准下适合用于开展射波刀脊柱临床计划的剂量验证工作。     

Familial alcoholism in manic-depressive (bipolar) disease

A previous analysis found a relatively high rate of alcoholism in a cohort of bipolar I subjects, and a trend for increased rates of alcoholism in relatives of subjects with both bipolar I disorder and alcoholism, compared to relatives of subjects with bipolar I disorder and no alcoholism. The sample of subjects with bipolar I disorder has been enlarged through continued follow-up, permitting new analyses to address the association and heritability of bipolar I disorder with alcoholism. Probands with bipolar I disorder were followed for 10 years as part of the NIMH Collaborative Depression Study. The rate of alcoholism in relatives of probands with both bipolar I disorder and alcoholism was compared to the rate of alcoholism in relatives of probands with bipolar disorder and no alcoholism. The prevalence of alcoholism in relatives of subjects with bipolar I disorder was compared to the rate of alcoholism in relatives of control subjects. Relatives of probands with bipolar I disorder showed a higher rate of alcoholism than relatives of controls. Relatives of probands with bipolar I disorder and alcoholism showed a higher rate of alcoholism than relatives of probands with bipolar I disorder without alcoholism. These data suggest that familial alcoholism may contribute to a vulnerability to bipolar I disorder, and that there is a shared heritability for the two disorders. © 1996 Wiley-Liss, Inc.     

A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents

Clinical laboratories are recognizing the importance of implementing sensitive and specific molecular diagnostic tests. However, widespread adoption of these tests requires simplified workflows without requiring expensive supporting instrumentation. To enable microarray-based analysis to meet these requirements, we describe a valveless flow cell for disposable use that supports PCR coupled with microarray hybridization in the same chamber. The flow cell assembly consists simply of double-faced tape, a plastic microarray substrate, an absorbent, and a commercially-available hydrophilic thin film. The simple construction lends itself to low-cost and ease of manufacturing, yet several features reduce the complexity of the standard microarray workflow. First, there is no requirement for custom instrumentation. Second, the hydrophilic thin film allows uniform filling of a microfluidic chamber. Third, a geometric capillary stop design confines liquid to the microarray chamber during PCR, and thus eliminates the need for a valve or hydrophobic surface treatment. And fourth, imbibition drives the uniform removal of liquid reagents from the array chamber. Three hundred genomic copies of methicillin-resistant Staphylococcus aureus (MRSA) are detected in a flow cell with gel drop microarrays printed on an unmodified plastic substrate. This sensitivity is shown to be comparable to conventional methods (i.e., PCR in a tube, with separate hybridization in a microarray chamber, where amplicon is exposed to the workspace before and after hybridization). However, the flow cell combines these multiple steps into a simple, compact workflow without the need for complex valves or custom instrumentation and is less susceptible to contamination of the workspace than conventional methods because the amplicon is confined to the device.     

High-frequency detection of deletions and variable rearrangements at the ornithine transcarbamylase (OTC) locus by oligonucleotide array CGH

Ornithine transcarbamylase (OTC) deficiency is an X-linked inborn error of metabolism characterized by impaired synthesis of citrulline from carbamylphosphate and ornithine. Previously reported data suggest that only approximately 80% of OTC deficiency (OTCD) patients have a mutation identified by OTC gene sequencing.To elucidate the molecular etiology in patients with clinical signs of OTCD and negative OTC sequencing, we subjected their DNA to array comparative genomic hybridization (aCGH) using a custom-designed targeted 44 k oligonucleotide array. Whenever possible, parental DNA was analyzed to determine the inheritance or to rule out copy number variants in the OTC locus.DNA samples from a total of 70 OTCD patients were analyzed. Forty-three patients (43/70 or 61.5%) were found to have disease-causing point mutations in the OTC gene. The remaining 27 patients (27/70 or 38.5%) showed normal sequencing results or failure to amplify all or part of the OTC gene. Among those patients, eleven (11/70 or 15.7%) were found to have deletions ranging from 4.5 kb to 10.6 Mb, all involving the OTC gene. Sixteen OTCD patients (16/70 or 22.8%) had normal sequencing and oligoarray results. Analysis of the deletions did not reveal shared breakpoints, suggesting that non-homologous end joining or a replication-based mechanism might be responsible for the formation of the observed rearrangements.In summary, we demonstrate that approximately half of the patients with negative OTC sequencing may have OTC gene deletions readily identifiable by the targeted oligonucleotide-based aCGH. Thus, the test should be considered in OTC sequencing-negative patients with classic symptoms of the disease.     

Serological markers for detection of cancer (Review)

We reviewed the effectiveness of commercial serological markers for the early detection of cancer and monitoring cancer patients. Parameters, such as specificity and variability of tumor markers were compared with a new approach for cancer detection which was recently developed in our laboratory. We demonstrate that the absence of tumor specificity and the extremely high variability of tumor markers are the reason that none of them can be used for early cancer diagnosis. We developed a method for the isolation of tumor-associated antigens (TAA) and found that two soluble low-molecular mass 66 and 51 kDa proteins could be isolated from the blood of cancer patients. The first protein, albumin, belongs to a group of heat-shock proteins (HSP), while the second is connected with TAA. The progress in cancer is characterized by a relative decrease in the concentration of HSP and a high increase in blood levels of TAA. Our method was shown to be highly sensitive and specific for the early detection of different types of cancer, such as of the colon, uterus, ovary, and breast, as well as melanoma.     

Bioassay for the detection of hydroxamate-iron-chelators (Aerobactin)

Different Salmonella strains were tested for aerobactin production in a hydroxamate-bioassay with the aerobactin indicator strain E. coli LG 1522. The majority of hospital strains of Salmonella typhimurium produce hydroxamate siderophore. On the other hand S. typhimurium strains belonging to phage type n. c. 1/72/n. c., biochemical type b from human and animal sources, were unable to produce this siderophore. Serotypes other than S. typhimurium for example the multiresistent S. wien hospital strains, which were isolated in western europe and in the GDR, can excreate hydroxamate siderophore. Plasmids pIE 528 and pIE 5 234 isolated from Salmonella hospital strains produce hydroxamate siderophore in the enterobactin negative Salmonella typhimurium-strain enb-7. Thus, the hydroxamate bioassay may be a useful supplementary test for epidemiological strain characterization.     

Synergetic chemiluminescence and label-free dual detection for developing a hepatitis protein array

A dual detection system for protein arrays is presented that combines label-free detection by optical interference with chemiluminescence. A planar protein array that targets hepatitis B surface antigen is developed. Surface densities for individual antibody spots are quantitated using optical interference prior to use. Target binding (10 ng/ml) is detected label-free. Target binding (1 ng/ml) is detected by both optical interference and chemiluminescence with the inclusion of secondary antibodies. Binding results using both methods are found to be directly proportion to the capture probe density measured initially. The dual detection system provides the analytical utility of optical interference detection with the established clinical utility of chemiluminescence detection.     

Design and validation of a low density array (Nosochip) for the detection and identification of the main pathogenic bacteria and fungi responsible for nosocomial pneumonia

The aim of this study was to be able to amplify and to detect on one array 27 different etiologic agents found in nosocomial pneumonia, some being phylogenetically closely related and others very distant. The assay is based on the use of consensus primers combined with the identification of the resulting amplicons by hybridization on specific capture probes present on an array. Three genes were necessary in order to cover the different pathogens. We took a redundancy of at least two positive spots to confirm the identity of each species. Each probe was present in triplicate on the array. The detection limit was between 10 and 1,000 DNA copies in the assay depending on the bacteria and the probe. The assay was also specific when tested both on reference collection strains corresponding to the 27 species of interest and on 57 other bacterial species of the normal human flora. Accuracy of the assay was assessed on 200 clinical isolates and some polymorphisms were indeed observed for 5 species. Effectiveness of the assay was preliminarily validated on 25 endotracheal aspirates and sputum samples, and the results were in accordance either with the cell culture or with the sequencing. Polybacterial infections were well detected in three samples. The results show that a combination of appropriate polymerase chain reaction (PCR) and redundancy of signals on the array allows specific screening of bacteria belonging to different species and genus and even fungi. The results open the way for a possible molecular detection of bacteria in the clinical diagnostic setting.