UCLA

Nucleic acid-based detection can be applied broadly to detect pathogens in, for example, clinical samples, livestock, food and water. The recent COVID-19 pandemic once again emphasized the importance of point-of-care (POC) diagnosis, which is rapid, inexpensive and reliable, to defend against public health threats. Currently, nucleic acid amplification tests (NAATs) rely on polymerase chain reaction (PCR) and other amplification methods that makes them more complex, less portable, dependent on perishable reagents and less suitable as POC tests. With the goal to develop portable, rapid and inexpensive diagnostic devices, we have been working on the further evolution of the previously reported nanopore-based, nucleic acid detection platform developed at UCLA. The detection scheme utilizes charge neutral peptide nucleic acid (PNA) probes conjugated to carboxyl-functionalized microbeads for sequence-specific nucleic acid (NA) detection. The hybridization event with target NA introduces negative charge to the PNA-bead complex which results in electrophoretic mobility. The negatively charged PNA-bead complex after target NA hybridization is directed by an electric field to a nanopore of smaller diameter in a micron-thick glass membrane where it blocks at least partially the ionic current flow thereby providing a sustained current drop which serves as the detection signal. In order to demonstrate the potential broad application for detection of pathogens in clinical samples, both Neisseria gonorrhoeae and Chlamydia trachomatis spiked in human urine were detected at 10-100 CFU/mL against a background of representative bacterial flora. High sensitivities and specificities of ~95% were demonstrated and no false positives were observed with the representative bacterial flora which served as a control. An improvement of overall detection time was achieved by integrating the nanopore detector, which consists of a glass chip with an etched membrane and nanopore, with the lateral flow assay (LFA) format. The extracted NA sample flows along an LFA membrane by surface-tension driven capillary flow and hybridizes with a preloaded deposit of neutral PNA-beads. Driven by an external electric field, the negatively charged bead with hybridized NA successfully blocks the smaller nanopore integrated on the LFA chip and generates a detection signal. Therefore, the overall detection time is reduced significantly without the need for complex pumps or valves. Detection of 10 aM E. coli 16S rRNA against 10 fM P. putida 16S rRNA within 15 minutes was demonstrated successfully using the nanopore integrated into the LFA format. Further demonstrations with complex clinical samples and integration with simpler NA extraction schemes may lead to broad application for disease diagnosis at the POC.The study of neurological disorders and the deciphering of complex behaviors rely on the ability to monitor neuronal processes including inter-neuronal chemical signaling. Compared to extensively developed electrophysiological recordings, the detection of neurotransmitter release events in near-real time is far less advanced and remains limited in application. Neuroprobes with high spatiotemporal resolution would enable establishment of the relationships between neurotransmitter release events and subsequent behaviors. Our lab previously has reported glutamate (Glut) microbiosensors that can selectively detect glutamate against a background of electroactive interferents including dopamine (DA) and ascorbic acid (AA). In order to achieve better sensitivity and spatiotemporal resolution, further development could be achieved by optimizing the enzyme deposition technique and the thicknesses of the deposited enzyme layer and permselective polymer coatings. Microcontact printing (μCP) has been adopted as a direct method to deposit arrays of proteins on silicon or glass substrates without compromising the activity of the proteins. By incorporating a specially designed polymer with choline oxide to create a μCP “ink” led to improved choline sensor performance including a sensitivity of 639 � 96 nA μM-1 cm-2 (pH 7.4; n = 4). Guided by simulations performed with a detailed mathematical model, choline sensors were constructed with exhibited sensitivity and response time of 660 � 40 nA μM−1 cm−2 at 37 �C and 0.36 � 0.05 s respectively. With assistance of microcontact printing to selectively transfer different enzymes onto specific microelectrodes in an array on 100-um-width microprobes, a demonstration of an implantable multifunctional neural microprobe for simultaneous multi-analyte sensing and chemical delivery has been achieved.