Bio-Inspired Fluorescent Carbon Dots as Probes for Rapid Detection of Bacteria in Physiological Samples

细菌感染对公共卫生和经济有很大的影响。虽然目前大多数病例可以用抗生素治疗，但这样做需要及早发现细菌感染病例，以便用正确的药物治疗，同时确保不不必要地使用抗生素。随着抗生素耐药性的增长，我们正确使用这些药物变得至关重要。目前，通常需要从患者样本（如尿液）中培养生物体，这一过程需要18个小时或更长时间，才能识别出特定的感染细菌。一种能够快速检测这些样本中细菌的存在，并识别出存在哪种细菌的设备，不需要这个生长步骤，将使医生能够迅速做出明智的决定，决定何时需要抗生素治疗，以及应该使用哪种药物。在这里，我们建议开发和评估一种技术，以确定细菌在病人的样本。我们将结合一系列新颖的化学探针（荧光碳点，FCDs），这些探针可以附着在细菌上使它们发出荧光，与我们的工业合作伙伴FluoretiQ有限公司开发的超灵敏量子光子传感器（QPS）相结合，该传感器能够检测患者样本中的这些荧光细菌。为了识别单个种类的细菌，我们将把特定的糖（聚糖）附着在fcd表面，利用不同的细菌识别特定的糖分子作为其与宿主细胞结合过程的一部分这一事实。我们的试验是围绕引起尿路感染的大肠杆菌进行的，因为这些是给NHS实验室带来高工作量的常见疾病（我们的临床合作伙伴每天处理多达1000份尿液样本），如果处理不当可能导致严重的疾病，如败血症。我们将通过在实验室评估特定细菌是否能与特定的聚糖- fcds结合来测试这种方法。第二个系列的实验室实验将寻求复制患者样本，方法是将来自患者的细菌和培养的人类细胞悬浮在模拟人类尿液组成的液体培养基中，并测试在这些条件下聚糖- fcds是否与细菌结合。最后，在临床微生物学家的支持下，我们将测试聚糖- fcd /QPS方法是否可以检测和鉴定人类患者尿液样本中的细菌，并与目前使用的方法进行比较，评估其有效性。作为未来的用户，他们还将帮助我们优化方法和相关仪器，以确保它可以在临床实验室中轻松使用，并就如何确保我们的方法可以针对适当的比较器进行验证并证明符合NHS质量管理体系提供指导。同时，我们将测试聚糖- fcds是否可以作为细菌感染新疗法的基础。我们已经证明FCDs可以与细菌结合并进入细菌；初步实验表明，它们还可以在一个依赖光的过程中杀死细菌。因此，我们将研究我们的修饰聚糖- fcds是否保留了杀死细菌的能力，以及这种杀死是否仅针对特定表面糖所针对的物种。我们还将把抗生素附着在fcd表面，以测试这是否代表了一种将药物输送到特定细菌的方法，其中许多细菌由于药物无法进入细菌细胞而难以用抗生素杀死。该项目将确定在临床微生物学实验室中，聚糖- fcds是否可以形成一种快速检测患者样本中感染细菌的方法的基础，以及这些方法是否也可以用于提高抗生素对许多这些微生物的有效性。在这样做的过程中，我们还将开发合成复杂糖分子的新方法，这些方法可能应用于包括药物和疫苗开发在内的多个其他研究领域。

Bacterial infections have great public health and economic impact. While at present most can be treated with antibiotics, doing so requires cases of bacterial infections to be recognised early so that they can be treated with the right drugs, while ensuring that antibiotics are not given unnecessarily. With the growth in antibiotic resistance, it is becoming essential that we use these drugs appropriately. At present growth of organisms from patient samples (e.g. urine), a process which takes 18 hours or more, is usually required before specific infecting bacteria can identified. A device able to rapidly detect the presence of bacteria in such samples, and identify which species are present, without this growth step would enable doctors to make rapid and informed decisions about when antibiotic treatment is necessary and which drug should be used.Here we propose to develop and evaluate a technology for identifying bacteria in patient samples. We will combine a novel series of chemical probes (fluorescent carbon dots, FCDs) that can attach to bacteria to make them fluorescent, with an ultra-sensitive quantum photonic sensor (QPS) developed by our industrial partner, FluoretiQ Ltd., that is able to detect these fluorescent bacteria in patient samples. In order to identify individual species of bacteria we will attach specific sugars (glycans) to the surface of FCDs, exploiting the fact that different bacteria recognise particular sugar molecules as part of the process of binding to the cells of their host. We base our trials around E coli bacteria causing urinary tract infections as these are common conditions that create high workloads for NHS laboratories (our clinical partner processes up to 1000 urine samples per day) and if improperly treated can lead to severe conditions such as sepsis.We will test this methodology by assessing in the laboratory whether specific bacteria can bind to specific glycan-FCDs. A second series of laboratory experiments will then seek to replicate patient samples by suspending bacteria derived from patients, and cultured human cells, in liquid media designed to mimic the composition of human urine and testing whether glycan-FCDs bind bacteria under these conditions. Finally, with support from clinical microbiologists, we will test whether the glycan-FCD/QPS method can detect and identify bacteria in urine samples from human patients and evaluate its effectiveness compared to methods currently in use. As future users they will also help us to optimise the method and associated instrumentation to ensure that this can be used easily in the clinical laboratory, and provide guidance on how to ensure that our method can be validated against appropriate comparators and demonstrated to comply with NHS quality management systems.In parallel we will test whether glycan-FCDs can be used as the basis for new treatments for bacterial infections. We have already demonstrated that FCDs can bind to and enter bacteria; preliminary experiments show that they can also kill bacteria, in a light-dependent process. Hence we will investigate whether our modified glycan-FCDs retain the ability to kill bacteria, and whether this killing is specific to the species targeted by the particular surface sugar. We will also attach antibiotics to the surface of FCDs to test whether this represents a method to deliver drugs to specific bacteria, many of which are difficult to kill with antibiotics because the drug is unable to enter the bacterial cell.The project will establish whether glycan-FCDs can form the basis of a rapid method for detecting infecting bacteria in patient samples in the clinical microbiology laboratory, and whether these can also be used to improve the effectiveness of antibiotics against many of these organisms. In so doing we will also develop new methods for synthesising complex sugar molecules that may be applied in multiple other research areas including drug and vaccine development.