Design of high stability thin-film transistor biosensor for the diagnosis of bladder cancer

Bladder cancer is the most common malignant tumor in the urinary system,with high morbidity,mortality and recurrence after surgery.However,current bladder cancer urine diagnosis methods are limited by the low accuracy and specificity due to the low abundance of bladder cancer biomarkers in the urine with complex biological environments.Herein,we present a high stability indium gallium zinc oxide field effect transistor(IGZO-FET) biosensor for efficient identification of bladder cancer biomarkers from human urine samples.The recognition molecular functionalized IGZO-FET biosensor exhibits stable electronic and sensing performance due to the large-area fabrication of IGZO thin-film FET.Owing to the excellent electrical performance of IGZO-FET,the IGZO-FET biosensor exhibits high sensitivity and extremely low detection limit(2.7 amol/L) towards bladder cancer biomarkers.The IGZO-FET biosensor is also able to directly detect bladder tumor biomarker in human urine with high sensitivity and specificity,and could differentiate bladder cancer patients' urine samples from healthy donors effectively.These results indicate that our designed high-performance biosensor shows great potential in the application of portable digital bladder cancer diagnosis devices.     

尿核基质蛋白-22和液基细胞学在血尿患者膀胱癌筛查中的应用

目的探讨尿核基质蛋白-22和液基细胞学在血尿患者膀胱癌筛查中的应用。方法选取2015年6月至12月在兴宁市中医医院收集的10例膀胱癌疑似患者,分别进行尿核基质蛋白-22检查与尿液基细胞学检。观察两种检查下患者的诊断结果。结果尿核基质蛋白-22的敏感度为80.2%,液基细胞学的敏感度为72.1%,两组之间作比较差异有统计学意义(P0.05)。尿核基质蛋白的特异度为71.6%,液基细胞学的特异度为98.6%,两组之间比较差异具有统计学意义(P0.05)。两种检查下患者阳性预测率及阴性预测率比较,差异显著。结论在血尿患者的膀胱癌筛查中,尿核基质蛋白22作为诊断膀胱癌的新肿瘤标记物对患者创伤性较小,灵敏度和特异性较高的诊断方法。而尿液基细胞学与核基质蛋白22联合使用可以有效提高对膀胱癌诊断的敏感程度。     

Analysis of VOCs in Urine Samples Directed towards of Bladder Cancer Detection

Bladder cancer is one of most common types of cancer diagnosed in the genitourinary tract. Typical tests are costly and characterized by low sensitivity, which contributes to a growing interest in volatile biomarkers. Head space solid phase microextraction (SPME) was applied for the extraction of volatile organic compounds from urine samples, and gas chromatography time of flight mass spectrometry (GC×GC TOF MS) was used for the separation and detection of urinary volatiles. A cohort of 40 adult patients with bladder cancer and 57 healthy persons was recruited. Different VOC profiles were obtained for urine samples taken from each group. Twelvecompounds were found only in the samples from theBC group.The proposed candidate biomarkers are butyrolactone; 2-methoxyphenol; 3-methoxy-5-methylphenol; 1-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)-2-buten-1-one; nootkatone and 1-(2,6,6-trimethyl-1-cyclohexenyl)-2-buten-1-one.Since most of the studies published in the field are proving the potential of VOCs detected in urine samples for the screening and discrimination of patients with bladder cancer from healthy, but rarely presenting the identity of proposed biomarkers, our study represents a novel approach.     

Design and Functionalization of a µPAD for the Enzymatic Determination of Nitrate in Urine

In this work, the design of a microfluidic paper-based analytical device (μPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination μPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed μPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14–1.0 mM. The designed μPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at −20 °C. The accuracy of the nitrate μPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.     

Construction of high stability indium gallium zinc oxide transistor biosensors for reliable detection of bladder cancer-associated microRNA

Bladder cancer is the most common malignant tumours with high morbidity, mortality and recurrence.However, currently developed detection methods for bladder cancer-associated urine biomarkers are hindered by their extremely low abundance. Hence, the exploration of a highly sensitive and selective approach for the detection of trace bladder cancer-associated biomarkers in human urine is of vital importance for the diagnosis of bladder cancer. Herein, we developed a highly reliable indium gallium ...     

Elevated metallothionein-bound cadmium concentrations in urine from bladder carcinoma patients, investigated by size exclusion chromatography-inductively coupled plasma mass spectrometry

Cadmium is discussed as being involved in the development of transitional cell carcinoma (TCC) of the bladder and can be observed in urine of these patients. Investigations of urinary samples from bladder cancer patients and normal controls were carried out with special emphasis on metallothionein (MT)-bound cadmium.Compounds that are constituents of urine were separated in urine samples by means of size exclusion chromatography and cadmium was monitored continuously with a hyphenated inductively coupled plasma mass spectrometry (ICP-MS) system. MT-bound cadmium was quantified by peak area integration, taking into account the intensity of the rhodium signal which was added continuously before ICP-MS detection.The obtained results show that urinary cadmium is predominantly bound to the observed MT-fraction. The median of the MT-bound cadmium concentration in the control group was found to be 0.8 μg L⁻¹ whereas the cancer group has a median of 1.8 μg L⁻¹. The variance of the data in the cancer group is much higher than in the controls. However, the urinary MT-bound cadmium is significantly elevated in the cancer group; odds-ratio test: 7.11 (95% C.I.: 1.89-26.80), taking into account the total protein content.Due to the fact that only one main cadmium-containing fraction was observed, there is no necessity to separate the MT-fraction before cadmium determination in urine samples in future studies.     

Thread/paper‐ and paper‐based microfluidic devices for glucose assays employing artificial neural networks

This paper describes the fabrication of and data collection from two microfluidic devices: a microfluidic thread/paper based analytical device (μTPAD) and 3D microfluidic paper‐based analytical device (μPAD). Flowing solutions of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI), through each device, on contact with glucose, generated a calibration curve for each platform. The resultant yellow‐brown color from the reaction indicates oxidation of iodide to iodine. The devices were dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentration. A similar procedure, using an unknown concentration of glucose in artificial urine, is conducted and compared to the calibration curve to obtain the unknown value. Studies to quantify glucose in artificial urine showed good correlation between the theoretical and actual concentrations, as percent differences were ≤13.0%. An ANN was trained on the four‐channel CMYK color data from 54 μTPAD and 160 μPAD analysis sites and Pearson correlation coefficients of R = 0.96491 and 0.9739, respectively, were obtained. The ANN was able to correctly classify 94.4% (51 of 54 samples) and 91.2% (146 of 160 samples) of the μTPAD and μPAD analysis sites, respectively. The development of this technology combined with ANN should further facilitate the use of these platforms for colorimetric analysis of other analytes.     

Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

膀胱癌液体活检的研究进展

膀胱癌是一种高发病率和致死率的恶性肿瘤疾病,通常情况下在中后期才能被诊断出来,给患者带来了巨大的身心伤害。 膀胱镜检查是膀胱癌诊断的金标准,但这种方法具有一定的侵入性,并且在膀胱癌的早期诊断中,灵敏度和特异性较低,容易出现较高的假阳性率。 膀胱癌的发生会对血液和尿液的成分产生直接影响,因此非侵入性的液体活检将为膀胱癌的早期诊断带来新的检测方法。 本篇综述介绍了基于液体活检的膀胱癌诊断方法的发展进程。 首先,对膀胱癌的主要标志物进行了简单介绍。 其次,重点总结了以液体(如尿液和血液)为检测对象的膀胱癌诊断方法和诊断机制。 除此之外,我们对膀胱癌液体活检面临的机遇和挑战进行了阐述。 我们希望这篇综述将为膀胱癌的液体活检提供指导。     

Urinary protein profiling by liquid chromatography/tandem mass spectrometry: ADAM28 is overexpressed in bladder transitional cell carcinoma

Bladder cancer is the most common urological cancer with higher incidence rate in the endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to utilize the proteomic approach to establish urinary protein patterns of bladder cancer. The experimental results showed that most patients with bladder cancer had proteinuria or albuminuria. The urine arsenic concentrations of bladder cancer patients in BFD areas were significantly higher than those patients from non-BFD areas. In the proteomic analysis, the urinary proteome was identified by nano-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (nano-HPLC/ESI-MS/MS) followed by peptide fragmentation pattern analysis. We categorized 2782 unique proteins of which 89 proteins were identified with at least three unique matching peptide sequences. Among these 89 proteins, thirteen of them were not found in the control group and may represent proteins specific for bladder cancer. In this study, three proteins, SPINK5, ADAM28 and PTP1, were also confirmed by Western blotting and showed significant differential expression compared with the control group. ADAM28 may be used as a possible biomarker of bladder cancer.     

Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study’s results may support a better understanding of bladder cancer development and progression mechanisms.     

Methodology for rapid tritium determination in urine

Summary The present work proposes a methodology for a rapid determina-tion of the tritium content in urine. The urine is treated with active carbon and tritium is determined by liquid scintillation counting. Different kinds of active carbon were experimented for the metabolites removal from urine samples. The counting efficiency was calculated by a quench curve using yellow food dye as a quenching agent. It was obtained a procedure which maintained a good reproducibility, low uncertainty and detection limit of 7 Bq/l (10 ml of urine, 120 minutes of counting time and efficiency of 23.9%).     

Determination of total oncopterin,neopterin and biopterin in human urine by high performance liquid chromatography with solid phase extraction

A new, sensitive method for the determination of oncopterin, biopterin, and neopterin in human urine has been developed using SPE with 6,7‐dimethylpterin as internal standard and gradient HPLC with fluorescence detection. SPE was tested for the pre‐treatment of urine samples on different types of sorbents (strong ion exchange resins, polar adsorbents, and reversed‐phase sorbents). RP‐SPE with subsequent evaporation of eluate has been found to be the most appropriate. The extraction efficiency exceeded 95% for all selected pterins. The extracted pterins were subsequently analyzed on a Purospher RP‐18 RP column. The LOD of oncopterin was 1.43 nmol/L of urine. The intra‐day and inter‐day imprecision at a physiological oncopterin concentration never exceeded 10%. The potential of this method was tested using urine samples of healthy volunteers and cancer patients without methotrexate therapy.     

Molecular genetic diagnosis of de novo and recurrent bladder cancer

Bladder cancer, the second most common urological tumor, is usually diagnosed by endoscopy and biopsy of the lower urinary tract. However, this procedure is expensive, can cause discomfort to the patient and is a source of infection. Commercially available diagnostic systems measure protein byproducts of bladder carcinoma in voided urine; their sensitivity is only between 60-80%. Polymerase chain reaction (PCR)-based microsatellite analysis of the urine sediment (MAUS) is a noninvasive, inexpensive and easily performed analytical method which was introduced in the de novo diagnosis and follow-up of bladder cancer. By utilizing the PCR with 20 polymorphic microsatellite markers on different chromosomes and separating PCR products by electrophoresis on 7% denaturing polyacrylamide-formamide-urea slab gels, a 91% diagnostic sensitivity could be achieved. In order to minimize costs and analysis time, the separation and detection of PCR products was carried out by capillary array electrophoresis and two-color fluorescent primer labeling/laser beam detection in another study. The accuracy of both methods was the same. In either detection system, MAUS is an accurate and promising tool in the noninvasive diagnosis of bladder cancer.     

In-tube solid-phase microextraction with poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary for direct high-performance liquid chromatographic determination of ketamine in urine samples

Ketamine was used for anaesthesia originally but has emerged as an abused drug in recent years. The prevalence of ketamine abuse demands a direct and rapid determination method. It is known that in-tube solid phase microextraction (in-tube SPME) can perform extraction with a capillary linked directly to a HPLC system, providing an automated and accurate extraction procedure. In this paper, an in-tube SPME coupled to HPLC method was developed for the determination of ketamine in urine samples with a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column as the extraction phase, which is expected to provide higher extraction efficiency than open tubular capillaries. After optimizing the extraction conditions, ketamine was extracted directly from urine samples in a wide dynamic linear range of 50-10,000 ng mL(-1), with the detection limit obtained as 6.4 ng mL(-1). The intra-day and inter-day precision for the method was 1.6% and 1.7%, respectively. The urine samples from suspect addicts have been successfully analyzed within 20 min. The re-usability of the monolithic column was also confirmed as no decrease of the extraction efficiency was shown after urine extraction.     

Evaluation of a gas sensor array and pattern recognition for the identification of bladder cancer from urine headspace

Previous studies have indicated that volatile compounds specific to bladder cancer may exist in urine headspace, raising the possibility that headspace analysis could be used for diagnosis of this particular cancer. In this paper, we evaluate the use of a commercially available gas sensor array coupled with a specifically designed pattern recognition algorithm for this purpose. The best diagnostic performance that we were able to obtain with independent test data provided by healthy volunteers and bladder cancer patients was 70% overall accuracy (70% sensitivity and 70% specificity). When the data of patients suffering from other non-cancerous urological diseases were added to those of the healthy controls, the classification accuracy fell to 65% with 60% sensitivity and 67% specificity. While this is not sufficient for a diagnostic test, it is significantly better than random chance, leading us to conclude that there is useful information in the urine headspace but that a more informative analytical technique, such as mass spectrometry, is required if this is to be exploited fully.     

Direct determination of pregabalin in human urine by nonaqueous CE‐TOF‐MS

Determination of pregabalin in urine samples was carried out by nonaqueous CE with TOF‐MS via ESI, with a mixture of 10 mM ammonium formate and 0.05% acetic acid in methanol. By using TOF‐MS, accurate mass information was obtained, thus causing a great improvement in qualitative ability. In order to avoid ionic suppression, urine samples dilution 1:10 was used. This was the only treatment to urine samples before the injection. Despite this dilution, the detection limit was as low as 0.03 μg/mL for pregabalin. The method was validated with respect to accuracy, precision, and linearity, LOD, and LOQ. This method was applied to the analysis of urine samples from seven different cancer patients undergoing treatment with pregabalin. The developed method may find wide application for the routine determination of pregabalin in biological samples in order to establish a more efficient and safe dosage.     

Simultaneous determination of pseudouridine and creatinine in untreated urine by ion-pair liquid chromatography with diode-array ultraviolet detection

A simple procedure for the simultaneous determination of pseudouridine and creatinine in urine using ion-pair high-performance liquid chromatography with ultraviolet detection is described. It consists of simply diluting the filtered urine with mobile phase (1:20) followed by direct chromatographic injection. A single analysis takes only 10 min. This method has been applied to the analysis of urine samples from normal donors and patients with different types of cancer. The mean values, means, of the peak-area ratio of pseudouridine to creatinine were 61.79.10(-3) and 81.92.10(-3) for male and female normal donors, respectively. Out of twenty-five urine samples of patients with cancer examined, nineteen (all the forteen males included) had values higher than means + 2 sigma.     

Mixed hemimicelles SPE based on CTAB-coated Fe3O4/SiO2 NPs for the determination of herbal bioactive constituents from biological samples

In this paper, a solid-phase extraction (SPE) method based on mixed hemimicelles of cetyltrimethyl ammonium bromide (CTAB) on silica-coated magnetic nanoparticles (MNPs) is developed for extraction and preconcentration of compounds from the biological samples. We selected rhein and emodin which are the major active anthraquinones of rhubarb as model analytes. A high performance liquid chromatography-fluorescence detection (HPLC/FLD) method was developed for the determination of rhein and emodin in urine and serum samples. The main factors influencing the extraction efficiency including the amount of surfactant, the concentration of MNPs, the shaking time and the desorption ability of organic solvents were investigated and optimized. No interferences were caused by proteins or endogenous compounds in urine and serum samples. Good linearities (r² > 0.9995) for all calibration curves were obtained, and the limits of detection (LODs) for rhein and emodin were 0.2 and 0.5 ng/mL in urine samples and 7 and 10 ng/mL in serum samples, respectively. Satisfactory recoveries (92.76-109.90% and 97.53-107.72% for rhein and emodin) in the biological matrices were achieved.     

Determination of homogentisic acid in urine for diagnosis of alcaptonuria: Capillary electrophoretic method optimization using experimental design

Homogentisic acid (HGA) is a diagnostic metabolite that accumulates in the urine and tissues of patients with alkaptonuria which is a rare autosomal recessive disease. HGA is a specific metabolite in urine and serum, which is used for diagnosis of alkaptonuria. This study presents an inexpensive and easy capillary electrophoretic method for the quantitative determination of HGA in urine samples. The method was optimized using full factorial experimental design. The optimal separation electrolyte and separation voltage were revealed as 45 mmol/L phosphate buffer at pH 7.0 and 22 kV, respectively. Under these conditions the presence of HGA was detected in 6 min. Repeatability of migration times and corrected peak areas of HGA (as RSD) were 0.37 and 1.99, respectively. The detection limit was 0.56 μg/mL, 3 times of the average noise, and the quantification limit was 1.85 μg/mL, 10 times the average noise for HGA. Urine samples were directly injected to the capillary without any pretreatment step.