RESUMO
Rapid and accurate diagnosis of bacterial infections resistant to multiple antibiotics requires development of new bio-sensors for differentiated detection of multiple targets. This work demonstrates 7x multiplexed detection for antibiotic-resistance bacterial screening on an optofluidic platform. We utilize spectrally multiplexed multi-spot excitation for simultaneous detection of nucleic acid strands corresponding to bacterial targets and resistance genes. This is enabled by multi-mode interference (MMI) waveguides integrated in an optofluidic device. We employ a combinatorial three-color labeling scheme for the nucleic acid assays to scale up their multiplexing capability to seven different nucleic acids, representing three species and four resistance genes.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Enterobacter aerogenes/genética , Escherichia coli/genética , Klebsiella pneumoniae/genética , Técnicas de Amplificação de Ácido Nucleico , Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Enterobacter aerogenes/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana , Dispositivos ÓpticosRESUMO
Nucleic acids and proteins are the two most important target types used in molecular diagnostics. In many instances, simultaneous sensitive and accurate detection of both biomarkers from the same sample would be desirable, but standard detection methods are highly optimized for one type and not cross-compatible. Here, we report the simultaneous multiplexed detection of SARS-CoV-2 RNAs and antigens with single molecule sensitivity. Both analytes are isolated and labeled using a single bead-based solid-phase extraction protocol, followed by fluorescence detection on a multi-channel optofluidic waveguide chip. Direct amplification-free detection of both biomarkers from nasopharyngeal swab samples is demonstrated with single molecule detection sensitivity, opening the door for ultrasensitive dual-target analysis in infectious disease diagnosis, oncology, and other applications.
RESUMO
Bacterial pathogens resistant to antibiotics have become a serious health threat. Those species which have developed resistance against multiple drugs such as the carbapenems, are more lethal as these are last line therapy antibiotics. Current diagnostic tests for these resistance traits are based on singleplex target amplification techniques which can be time consuming and prone to errors. Here, we demonstrate a chip based optofluidic system with single molecule sensitivity for amplification-free, multiplexed detection of plasmids with genes corresponding to antibiotic resistance, within one hour. Rotating disks and microfluidic chips with functionalized polymer monoliths provided the upstream sample preparation steps to selectively extract these plasmids from blood spiked with E. coli DH5α cells. Waveguide-based spatial multiplexing using a multi-mode interference waveguide on an optofluidic chip was used for parallel detection of three different carbapenem resistance genes. These results point the way towards rapid, amplification-free, multiplex analysis of antibiotic-resistant pathogens.
Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/farmacologia , Carbapenêmicos , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genéticaRESUMO
Nanopore-based single nanoparticle detection has recently emerged as a vibrant research field with numerous high-impact applications. Here, we introduce a programmable optofluidic chip for nanopore-based particle analysis: feedback-controlled selective delivery of a desired number of biomolecules and integration of optical detection techniques on nanopore-selected particles. We demonstrate the feedback-controlled introduction of individual biomolecules, including 70S ribosomes, DNAs and proteins into a fluidic channel where the voltage across the nanopore is turned off after a user-defined number of single molecular insertions. Delivery rates of hundreds/min with programmable off-times of the pore are demonstrated using individual 70S ribosomes. We then use real-time analysis of the translocation signal for selective voltage gating of specific particles from a mixture, enabling selection of DNAs from a DNA-ribosome mixture. Furthermore, we report optical detection of nanopore-selected DNA molecules. These capabilities point the way towards a powerful research tool for high-throughput single-molecule analysis on a chip.
Assuntos
Dispositivos Lab-On-A-Chip , Nanoporos , Dispositivos Ópticos , Imagem Individual de Molécula/instrumentação , DNA , Escherichia coli , RibossomosRESUMO
We use optical trapping to deliver molecular targets to the vicinity of a nanopore for high-throughput single molecule analysis on an optofluidic chip. DNA detection rates increase over 80× to enable detection at attomolar concentrations.
RESUMO
An automated microfluidic sample preparation multiplexer (SPM) has been developed and evaluated for Ebola virus detection. Metered air bubbles controlled by microvalves are used to improve bead-solution mixing thereby enhancing the hybridization of the target Ebola virus RNA with capture probes bound to the beads. The method uses thermally stable 4-formyl benzamide functionalized (4FB) magnetic beads rather than streptavidin coated beads with a high density of capture probes to improve the target capture efficiency. Exploiting an on-chip concentration protocol in the SPM and the single molecule detection capability of the antiresonant reflecting optical waveguide (ARROW) biosensor chip, a detection limit of 0.021pfu/mL for clinical samples is achieved without target amplification. This RNA target capture efficiency is two orders of magnitude higher than previous results using streptavidin beads and the limit of detection (LOD) improves 10×. The wide dynamic range of this technique covers the whole clinically applicable concentration range. In addition, the current sample preparation time is ~1h which is eight times faster than previous work. This multiplexed, miniaturized sample preparation microdevice establishes a key technology that intended to develop next generation point-of-care (POC) detection system.
Assuntos
Técnicas Biossensoriais/instrumentação , Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/virologia , Técnicas Analíticas Microfluídicas/instrumentação , RNA Viral/análise , Desenho de Equipamento , Doença pelo Vírus Ebola/diagnóstico , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Extração em Fase Sólida/instrumentação , Estreptavidina/químicaRESUMO
We have developed an optofluidic analysis system that processes biomolecular samples starting from whole blood and then analyzes and identifies multiple targets on a silicon-based molecular detection platform. We demonstrate blood filtration, sample extraction, target enrichment, and fluorescent labeling using programmable microfluidic circuits. We detect and identify multiple targets using a spectral multiplexing technique based on wavelength-dependent multi-spot excitation on an antiresonant reflecting optical waveguide chip. Specifically, we extract two types of melanoma biomarkers, mutated cell-free nucleic acids -BRAFV600E and NRAS, from whole blood. We detect and identify these two targets simultaneously using the spectral multiplexing approach with up to a 96% success rate. These results point the way toward a full front-to-back chip-based optofluidic compact system for high-performance analysis of complex biological samples.
RESUMO
The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.
Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , HumanosRESUMO
Urinary fistula is an acknowledged complication of partial nephrectomy. We describe a case of a urinary fistula that failed to respond to conventional treatment and the subsequent use of percutaneous Hystoacryl glue to achieve its resolution.
Assuntos
Nefropatias/terapia , Nefrectomia/efeitos adversos , Adesivos Teciduais/uso terapêutico , Fístula Urinária/terapia , Acrilatos/uso terapêutico , Carcinoma de Células Renais/cirurgia , Feminino , Humanos , Nefropatias/diagnóstico por imagem , Nefropatias/etiologia , Neoplasias Renais/cirurgia , Pessoa de Meia-Idade , Radiografia , Fístula Urinária/diagnóstico por imagem , Fístula Urinária/etiologia , Urinoma/diagnóstico por imagem , Urinoma/etiologiaRESUMO
BACKGROUND: A representative sample of the British population was interviewed to identify the factors associated with sunburn and intention to tan, and the source from which people obtained their information about the risks of sun exposure. METHODS: As part of its rolling Omnibus survey, the Office for National Statistics interviewed a randomly selected representative sample of 1858 adults (aged 16 or over) in Great Britain in October 1996. The analysis and interpretation of the resulting data form the basis of this study. RESULTS: The response rate was just below 70 per cent. Forty-three per cent of men and 35 per cent of women reported sunburning in the 12 months to October 1996. Factors associated with sunburn were: being male, having a skin that tans with difficulty and being younger. Concerning 'intention to tan', 34 per cent of men and 39 per cent of women admitted trying. There were associations with having a skin that tans easily, and, less strongly, with being younger. There was a clear association between trying to tan and severity of sunburn. Most people identified television as their main source of information about the dangers of sun exposure, and the majority had seen the sunburn forecasts on weather reports. Comparison with a previous Omnibus sunburn survey from 1993 shows little change in the proportion of the population becoming sunburnt, but some increase in the proportion of men intending to tan. CONCLUSIONS: The data allow an insight into current sun-related behaviour and whether there has been a change since 1993. Health promotion campaigns warning about the risk from the sun may have increased awareness, but have thus far failed to reduce the proportion becoming sunburnt. It is time to re-evaluate the thinking behind such programmes.
Assuntos
Comportamentos Relacionados com a Saúde , Conhecimentos, Atitudes e Prática em Saúde , Queimadura Solar/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Coleta de Dados , Feminino , Humanos , Incidência , Serviços de Informação , Masculino , Pessoa de Meia-Idade , Classe Social , Medicina Estatal , Queimadura Solar/prevenção & controle , Queimadura Solar/psicologia , Protetores Solares/administração & dosagem , Reino Unido/epidemiologiaRESUMO
A series of 40 patients took part in a double-blind, placebo-controlled trial of indoramin in prostatic bladder outflow obstruction. Patients were assessed clinically and urodynamically before and after 4 weeks' treatment. Significant improvement was seen in nocturia, volume voided, flow rates and residual urine. The drug was well tolerated, although 7 patients on treatment and 7 on placebo noted side effects. These results suggest that indoramin may be a useful agent in the symptomatic management of bladder outflow obstruction.
Assuntos
Indoramina/uso terapêutico , Hiperplasia Prostática/complicações , Obstrução do Colo da Bexiga Urinária/tratamento farmacológico , Método Duplo-Cego , Humanos , Masculino , Bexiga Urinária/fisiopatologia , Obstrução do Colo da Bexiga Urinária/etiologia , Obstrução do Colo da Bexiga Urinária/fisiopatologia , UrodinâmicaRESUMO
Two hundred and forty four patients underwent either simultaneous bilateral inguinal hernia repair (n = 122) or unilateral inguinal hernia (n = 122) repair at a general hospital between January 1971 and December 1981. The two groups of patients were matched for age and sex. Both groups had a similar overall incidence of post-operative complications and in both groups the duration of post-operative stay and duration of operating time were similar. Chest infections developed in 12 patients after bilateral repair and in 3 patients after unilateral repair (P less than 0.02). All patients were assessed prospectively from 4 to 15 years after operation, when no significant difference in the number of recurrent hernias was found. Our results suggest that simultaneous bilateral inguinal herniorrhaphy is economical in terms of both operating time and duration of hospital stay, and that this economy is not bought at a cost of increased short term morbidity or long-term recurrence rate.
Assuntos
Hérnia Inguinal/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Estudos Prospectivos , RecidivaRESUMO
A retrospective study of 100 consecutive nephrectomy patients showed a 20% catheterisation rate. Of the 11 carried out post-operatively only 4 were for urinary retention. The post-operative catheterisation rate for males was 7.1% and for females 2%. The rate of post-operative retention was 4.4%, which is much lower than that reported from other units. This rate of post-operative catheterisation for retention is so low that in spite of the very mild increase in complications compared with intra-operative catheterisation, the practice of routine prophylactic catheterisation of nephrectomy patients is unnecessary and cannot be recommended.
Assuntos
Nefrectomia , Cateterismo Urinário , Retenção Urinária/prevenção & controle , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriúria/etiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Cuidados Intraoperatórios , Masculino , Pessoa de Meia-Idade , Cuidados Pós-Operatórios , Estudos Retrospectivos , Cateterismo Urinário/efeitos adversosRESUMO
OBJECTIVE: To assess the outcome of men presenting with lower urinary tract symptoms (LUTS) associated with large postvoid residual urine volumes (PVR). PATIENTS AND METHODS: The study included men presenting with LUTS and a PVR of > 250 mL who, because of significant comorbidity, a low symptom score or patient request, were managed conservatively and prospectively, and were followed with symptom assessment, serum creatinine levels, flow rates and renal ultrasonography. Patients were actively managed if there was a history of previous outflow tract surgery, prostate cancer, urethral strictures, neuropathy, elevated creatinine or hydronephrosis. In all, 93 men (mean age 70 years, range 40-84) with a median (range) PVR of 363 mL (250-700) were included in the study and followed for 5 (3-10) years. At presentation, the median maximum flow rate was 10.2 (3-30) mL/s and the voided volume 316 (89-714) mL. RESULTS: The measured PVR remained stable in 47 (51%), reduced in 27 (29%) and increased in 19 (20%) patients; 31 patients (33%) went on to transurethral resection of the prostate after a median of 30 (10-120) months, because of serum creatinine elevation (two), acute retention (seven), increasing PVR (eight) and worsening symptoms (14). Of 31 patients 25 were available for evaluation after surgery; their median PVR was 159 (0-1000) mL, flow rate 18.4 (4-37) mL/s and voided volume 321 (90-653) mL. Symptoms were improved in all but five men. There was no difference in initial flow rate, voided volume or PVR between those who developed complications or went on to surgery and those who did not. Urinary tract infections (UTIs) occurred in five patients and two developed bladder stones. CONCLUSIONS: Complications such as renal failure, acute retention and UTIs are uncommon in men with large, chronic PVRs. Conservative management for this group of patients is reasonable but outpatient review is prudent. There were no factors that could be used to predict those patients who eventually required surgery.
Assuntos
Hiperplasia Prostática/complicações , Neoplasias da Próstata/terapia , Retenção Urinária/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Hiperplasia Prostática/terapia , Ressecção Transuretral da Próstata/métodos , Resultado do Tratamento , Retenção Urinária/fisiopatologia , Micção/fisiologia , UrinaRESUMO
One hundred patients undergoing cholecystectomy underwent ultrasonography of the biliary tree on the day prior to surgery. At operation a per-operative cholangiogram was performed unless stones were palpable in the duct. Pre-operative biliary ultrasonography accurately identified dilatation of the common bile duct (sensitivity 96%, specificity 95%) but was less accurate at detecting common duct stones (sensitivity 36%, specificity 98%). Thirty three percent of patients with dilated ducts on ultrasound did not have stones in the duct, while 20% of patients with common duct stones had normal sized ducts. We conclude that ultrasonography alone cannot reliably select patients who require exploration of the common bile duct, or select patients for operative cholangiography. Although pre-operative demonstration of common bile duct dilation is an absolute indication for operative cholangiography, by itself it does not indicate the need for exploration.
Assuntos
Colangiografia , Colecistectomia , Ducto Colédoco/diagnóstico por imagem , Adulto , Idoso , Doenças do Ducto Colédoco/diagnóstico por imagem , Dilatação Patológica/diagnóstico por imagem , Cálculos Biliares/diagnóstico por imagem , Humanos , Período Intraoperatório , Pessoa de Meia-Idade , Cuidados Pré-Operatórios , UltrassonografiaRESUMO
OBJECTIVE: To assess the current efficacy and safety of definitive external beam radiotherapy (EBRT) in the treatment of invasive bladder transitional cell carcinoma (TCC) in a district general hospital with cancer-centre status. PATIENTS AND METHODS: The case notes of all patients with bladder TCC undergoing EBRT with curative intent over an 8-year period (1988-95) were reviewed. Additional missing outcome data were collected. RESULTS: In all, 120 patients (109 men; median age 70 years, range 34-90) underwent radical EBRT (40-65 Gy; fraction median=20) over the 8-year period. Staging, as assessed by examination under anaesthesia and computed tomography, was T1 in 16%, T2 in 43%, T3 in 38% and T4 in 3%. In 96 patients (80%) the tumour was poorly differentiated (G3). The overall morbidity at 12 months was 12%; proctitis occurred in nine patients (8%) and cystitis in five (4%). Sixty-seven patients (59%) developed a local recurrence and in 36 (30%) this was invasive. The overall median survival was 60 months. Thirty-three patients underwent salvage cystectomy with a subsequent median survival of 12.5 months. CONCLUSION: Modern radical multifraction EBRT in invasive bladder TCC has a low morbidity, with an overall median survival of 5 years.
Assuntos
Institutos de Câncer , Carcinoma de Células de Transição/radioterapia , Neoplasias da Bexiga Urinária/radioterapia , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Hospitais de Distrito , Hospitais Gerais , Humanos , Masculino , Auditoria Médica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Análise de SobrevidaRESUMO
OBJECTIVE: To assess the level and morphological distribution of cyclooxygenase (COX)-1 and -2 in human prostates and to determine any association with the Gleason grade of prostate cancer. Materials and methods The study comprised 30 samples from patients with benign prostatic hyperplasia (BPH) and 82 with prostate cancer. Immunohistochemistry was used to assess the expression of COX-1 and -2, and 13 samples were also assessed using immunoblotting (six BPH and seven cancers). RESULTS: For both BPH and prostate cancer, COX-1 expression was primarily in the fibromuscular stroma, with variable weak cytoplasmic expression in glandular/neoplastic epithelial cells. In contrast, COX-2 expression differed markedly between BPH and cancer. In BPH there was membranous expression of COX-2 in luminal glandular cells and no stromal expression. In cancer the stromal expression of COX-2 was unaltered, but expression by tumour cells was significantly greater (P = 0.008), with a change in the staining pattern from membranous to cytoplasmic (P < 0.001). COX-2 expression was significantly higher in poorly differentiated than in well differentiated tumours (P < 0.001). These results were supported by immunoblotting, which showed similar levels of COX-1 in both BPH and cancer, but four times greater expression of COX-2 in cancer than in BPH. CONCLUSION: This is the first study to assess the co-expression of COX-1 and COX-2 proteins in benign and malignant human prostates, and showed the induction and significantly greater expression of COX-2 in cancer, which was also associated with tumour grade. The regular use of nonsteroidal anti-inflammatory drugs is associated with a reduced incidence of cancers. The present results provide the basis for a potential role for COX-2 inhibitors in the prevention and treatment of prostate cancer.