An ultrasensitive and culture-free detection platform for microorganisms and protein biomarkers

S.-T. Yau
United States

Keywords: rapid diagnosis, culture free bacterial detection, rapid antibiotics susceptibility testing


State-of-the-art technologies for the diagnosis of bacterial infections are based on fluorescence, mass spectrometry or polymerase chain reaction and require positive blood culture, leading to a time-to-result of 18-72 hours. The long diagnosis time caused by culture results in the use of broad-spectrum antibiotics, which results in under treatment, harmful side effects and the prevalence of drug resistant microorganisms. The detection of protein biomarkers using enzyme-linked immunosorbent assay is known to provide unsatisfactory sensitivity. We have developed an electrochemical immunoassay platform. The platform detects a range of disease-related targets including bacteria, viruses, fungi and protein biomarkers. The analytical performance of the platform has been validated. The detected cancer biomarkers include CA 125, PSA in serum and AMACR, a novel biomarker for prostate cancer, in serum and urine. The PSA and AMACR detections were performed on the femto gram/mL level. A biomarker for traumatic brain injury was detected in serum on the 10 fg/mL level. The two detected bacteria are E. coli in milk, meat juice and whole human blood and Shigella in blood and stool. Because of the intrinsic amplification provided by the platform, the detection was performed directly in blood samples without culture, resulting in a significantly shortened assay time. We have demonstrated an 84-minute detection-identification of E. coli in spiked blood below the 10 CFU/mL level. Using wild-type and ampicillin resistant E. coli strains, we achieved a 204-minute AST without culture and bacteria isolation. Most recently, we have tested the platform with spiked and clinical blood samples. The bacteria used in these studies include E. coli, K. pneumonia, P. aeruginosa, N. gonorrhroae, S. aureus, MRSA and Streptococcus pneumoniae. The platform was used to perform detection-identification-AST directly on these samples on the 3-5 CFU/mL level. The rate of success for these tests was 100%. Some of the tests were performed in parallel with two state-of-the-art technologies, (i.e., MALDI-TOF and MicroScan). The platform results compare favorably with those obtained with these technologies. However, the platform produced results about 15-27 hours sooner. The results of simultaneous detection-identification show that the platform yielded similar bacteria concentration as the culture value in only 84 minutes while culture took 16 hours. The platform performed AST on clinical blood samples in only 144 minutes and the results were in agreement with those obtained with MicroSan. Here, the samples were exposed to antibiotics for only one hour before detection on the platform. This platform, compared with current detection methods, provides three distinctive advantages: (1) ultrasensitive detection of bacteria (3-5 CFU/mL) and biomarkers (fg/mL) in complex matrices, (2) rapid diagnosis (3.8 hours) of bacterial infections without performing culture, and (3) disposable, inexpensive tests. These features are to be compared with state-of-the-art technologies. The platform will make a paradigm shift in the diagnosis of bacterial infections. It will allow healthcare providers to use narrow-spectrum antibiotics early in the treatment to achieve enhanced outcome. It will provide early detection of diseases such cancer and brain injuries.