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Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis.

ABSTRACT: Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard.

SUBMITTER: Pan Y

PROVIDER: S-EPMC2946447 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis.

Pan Ying Y Sonn Geoffrey A GA Sin Mandy L Y ML Mach Kathleen E KE Shih Mei-Chiung MC Gau Vincent V Wong Pak Kin PK Liao Joseph C JC

Biosensors & bioelectronics 20100708 2

Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract in ...[more]

PMID: 20667707

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Project description:Palm kernel cake (PKC) is the solid residue following oil extraction of palm kernels and useful to fatten animals either as a single feed with only minerals and vitamins supplementation, or mixed with other feedstuffs such as corn kernels or soy beans. The occurrence of mycotoxins (aflatoxins, ochratoxins, zearalenone, and fumonisins) in feed samples affects the animal's health and also serves as a secondary contamination to humans via consumption of eggs, milk and meats. Of these, aflatoxin B? (AFB?) is the most toxically potent and a confirmed carcinogen to both humans and animals. Methods such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) are common in the determination of mycotoxins. However, these methods usually require sample pre-treatment, extensive cleanup and skilled operator. Therefore, in the present work, a rapid method of electrochemical immunosensor for the detection of AFB? was developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Multi-walled carbon nanotubes (MWCNT) and chitosan (CS) were used as the electrode modifier for signal enhancement. N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activated the carboxyl groups at the surface of nanocomposite for the attachment of AFB?-BSA antigen by covalent bonding. An indirect competitive reaction occurred between AFB?-BSA and free AFB? for the binding site of a fixed amount of anti-AFB? antibody. A catalytic signal based on horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H?O?) and 3,3',5,5'-tetramethylbenzidine (TMB) mediator was observed as a result of attachment of the secondary antibody to the immunoassay system. As a result, the reduction peak of TMB(Ox) was measured by using differential pulse voltammetry (DPV) analysis. Based on the results, the electrochemical surface area was increased from 0.396 cm² to 1.298 cm² due to the electrode modification with MWCNT/CS. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.0001 to 10 ng/mL with limit of detection of 0.1 pg/mL. Good recoveries were obtained for the detection of spiked feed samples (PKC, corn kernels, soy beans). The developed method could be used for the screening of AFB? in real samples.

Project description:BackgroundEarly availability of pathogen identification in urinary tract infections (UTIs) has critical importance in disease management. Metagenomic next-generation sequencing (mNGS) has the potential to transform how acute and serious infections are diagnosed by offering unbiased and culture-free pathogen detection. However, clinical experience with application of the mNGS test is relatively limited.MethodsWe therefore established a MinION-based mNGS pathogens diagnostic platform and evaluated its potential for clinical implementation in UTIs with clinical samples. 213 urine samples from patients with suspected UTIs were included and subjected to mNGS testing using the MinION platform. mNGS results were compared to the gold standard of clinical culture and composite standard of combining clinical testing, confirmatory qPCR testing, and clinical adjudication by doctors.ResultsThe mNGS exhibited a sensitivity of 81.4% and a specificity of 92.3%, along with a positive predictive value of 96.6%, a negative predictive value of 64.9%, and an overall accuracy of 84.4%, all of which were determined based on the gold standard of routine culture results. When assessed against the composite standard, the sensitivity and specificity both increased to 89.9% and 100%, respectively, while the accuracy rose to 92.4%. Notably, the positive predictive value and negative predictive value also saw improvements, reaching 100% and 76.8%, respectively. Moreover, this diagnostic platform successfully identified dsDNA viruses. Among the 65 culture-negative samples, the viral detection rate reached 33.8% (22/65) and was subsequently validated through qPCR. Furthermore, the automatic bioinformatics pipeline we developed enabled one-click analysis from data to results, leading to a significant reduction in diagnosis time.ConclusionThese results demonstrate that the pathogen detection performance of mNGS is sufficient for diagnostic testing in clinical settings. As the method is generally unbiased, it can improve diagnostic testing of UTIs and other microbial infections.

Dataset Information

Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis.

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Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis.

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