Biophysical Properties of Bifunctional Phage-Biosensor.

Biosensor research is a swiftly growing field for developing rapid and precise analytical devices for biomedical, pharmaceutical, and industrial use and beyond. Herein, we propose a phage-based biosensor method to develop a sensitive and specific system for biomedical detection. Our method is based on in vitro selected phages and their interaction with the targeted analytes as well as on optical properties that change according to the concentration of the model analyte. The green fluorescent protein (GFP) was chosen as our model analyte as it has its own well-known optical properties. Brilliant green was used as a reporter component for the sensor. Its presence enables a color intensity (absorbance) change when the analyte is present in the solution. Furthermore, the reporter dye functioned as a quencher for an additional lanthanide label in our assay. It mediated the specific phage-derived interference in the signal measured with the time-resolved luminescence. Most importantly, our results confirmed that the presented bifunctional phage with its liquid crystal properties enabled the measurement of GFP in a concentration-dependent, quantitative manner with a limit of detection of 0.24 µg/mL. In the future, our novel method to develop phage-based biosensors may provide highly sensitive and specific biosensors for biomedical or otherwise-relevant targets.

Detecting disease associated biomarkers by luminescence modulating phages.

Assessment of risk for a given disease and the diagnosis of diseases is often based on assays detecting biomarkers. Antibody-based biomarker-assays for diseases such as prostate cancer are often ambiguous and biomarker proteins are frequently also elevated for reasons that are unspecific. We have opted to use luminescence modulating phages for the analysis of known acute inflammatory response biomarker CRP (C-reactive protein) and biomarkers of prostate cancer in urine samples. Firstly, CRP was used to simulate the detection process in a controlled chemical environment. Secondly, we tried to classify more challenging lethal prostate cancer samples from control samples. Our unique method utilizes a special biopanning process in order to create special phages capable of capturing a dye necessary for detection and potential biomarkers. As the biomarker-molecules interfere with the phages, dye is repelled from the phage network resulting in an altered reporter luminescence. These changes can be observed with an absorbance reader and even with the naked eye. The simple method could present an alternative for screening of disease biomarkers. For prostate cancer urine samples, we achieved a sensitivity of 80% and specificity of 75% to detect Grade Group (GG) 4 and 5 prostate cancer.

Rapid time-resolved luminescence based screening of bacteria in urine with luminescence modulating biosensing phages.

Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages. Filamentous phage M13 was biopanned for binding luminescence quenching metal (copper) and further E. coli. The screening assay luminescence modulation was further enhanced by selecting right chemical environment for the functioning phage clones. Semi-specific interaction between phage, target bacteria and metal was detected by modulation in the signal of a weakly chelating, easily quenchable lanthanide complex. In the presence of the target pathogen, the phages collected quenching metal from solution to the bacterial surface changing the quenching effect on the lanthanide label and thus modulating the signal. Our method was compared with the bacterial culture data obtained from 70 patient samples. The developed proof-of-principle screening assay showed sensitivity and a specificity at the 90% mark when compared to culture method although some samples had high turbidity and even blood. The detection limit of E. coli was in the range of 1000-10 000 colony forming units/mL. Untreated urine sample was screened and time-resolved luminescence signal result was achieved within 10 min in a single incubation step.

Time-resolved fluorescence-based assay for rapid detection of Escherichia coli.

Fast and simple detection of pathogens is of utmost importance in health care and the food industry. In this article, a novel technology for the detection of pathogenic bacteria is presented. The technology uses lytic-specific bacteriophages and a nonspecific interaction of cellular components with a luminescent lanthanide chelate. As a proof of principle, Escherichia coli-specific T4 bacteriophage was used to infect the bacteria, and the cell lysis was detected. In the absence of E. coli, luminescent Eu(3+)-chelate complex cannot be formed and low time-resolved luminescence signal is monitored. In the presence of E. coli, increased luminescence signal is observed as the cellular contents are leached to the surrounding medium. The luminescence signal is observed as a function of the number of bacteria in the sample. The homogeneous assay can detect living E. coli in bacterial cultures and simulated urine samples within 25 min with a detection limit of 1000 or 10,000 bacterial cells/ml in buffer or urine, respectively. The detection limit is at the clinically relevant level, which indicates that the method could also be applicable to clinical settings for fast detection of urine bacteria.

Detection of melanoma-derived cancer-testis antigen CT16 in patient sera by a novel immunoassay.

Cancer-testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16-positive patients. A highly sensitive time-resolved fluorescence-based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon-Mann-Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.