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造成巨大影响。虽然目前大多数可以用抗生素治疗，但要做到这一点，就需要及早发现细菌感染病例，以便用正确的药物治疗，同时确保不使用不必要的抗生素。随着抗生素耐药性的增长，我们适当使用这些药物变得至关重要。目前，在鉴定特定感染细菌之前，通常需要从患者样品（例如尿液）中生长生物体，这一过程需要18小时或更长时间。一种能够快速检测此类样本中细菌的存在并识别存在哪些物种的设备，而无需此生长步骤，将使医生能够在需要抗生素治疗以及应该使用哪种药物时做出快速和明智的决定。我们将联合收割机与我们的工业合作伙伴FluoretiQ Ltd.开发的超灵敏量子光子传感器（QPS）结合，能够检测到病人样本中的荧光细菌。为了识别单个细菌物种，我们将特定的糖（聚糖）附着在FCD表面，利用不同细菌识别特定糖分子作为与宿主细胞结合过程的一部分的事实。我们的试验是围绕大肠杆菌引起的尿路感染进行的，因为这些是常见的情况，会给NHS实验室带来很大的工作量（我们的临床合作伙伴每天处理多达1000份尿样），如果处理不当，可能会导致严重的疾病，如败血症。我们将通过在实验室评估特定细菌是否可以与特定聚糖-FCD结合来测试这种方法。然后，第二系列实验室实验将试图通过将来自患者的细菌和培养的人类细胞悬浮在旨在模拟人类尿液成分的液体培养基中，并测试聚糖-FCD是否在这些条件下结合细菌来复制患者样本。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识别人类患者尿液样本中的细菌，并与目前使用的方法相比，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，以确保其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与适当的比较方法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-FCD是否可以用作细菌感染新治疗的基础。我们已经证明，FCD可以结合并进入细菌;初步实验表明，它们也可以在光依赖性过程中杀死细菌。因此，我们将研究我们的改性聚糖-FCD是否保留杀死细菌的能力，以及这种杀死是否对特定表面糖靶向的物种具有特异性。我们亦会将抗生素附着于食物包装上，以测试这是否能将药物输送至特定细菌，因为药物无法进入细菌细胞，所以抗生素很难将这些细菌杀死。该项目将确定聚糖食物包装能否成为临床微生物学实验室快速检测病人样本中感染细菌的方法的基础，以及这些是否也可以用来提高抗生素对许多这些生物体的有效性。在此过程中，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其他多个研究领域，包括药物和疫苗开发。

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.