Thrust 4: Translate diagnostic protocols to devices and instrumentation

The CPD will integrate technologies from the other Aims, to implement working systems, and to characterize their performance for impactful pathogens and sample types. Importantly, our “system integration” Aim, will require multidisciplinary teams from the other Aims to implement novel molecular biology methods with a novel microfluidic sample preparation technology, and a sensing transducer. Bringing them together results in a working system and assay protocol with unprecedented capabilities. The key engineering capabilities in this Aim are optomechanical engineering, flexible electronics, rapid prototyping (3D printing additive manufacturing), optomechanical design, and software. We anticipate devising systems for a variety of sensing environments that include wearables, point of care/handheld, point of care/desktop, and laboratory analysis systems. Some sensing environments may require integration with wireless technologies for networking of sensor arrays. This Aim will interact closely with the Machine Learning Aim, to provide data for meta-analysis.

Wearable pathogen sensors using screen-printed flexible electronics and wireless communication

Conventional pathogen detection methods utilize bulky and expensive analytical instruments that require professional operators in a laboratory setting. Due to pressing needs for rapid results and lower cost, point of care (POC) pathogen analysis is a key capability for low resource settings, agricultural applications, and timely quarantine decision-making. Our team has expertise in developing a variety of portable/wearable sensing systems, including those based upon electrochemistry, electrochemiluminescence and photoelectrochemistry. Using Bluetooth and other near field communications (NFC) standards, smartphones can connect with external sensors, enabling data transmission and signal processing. We demonstrated a smartphone-based electrochemical sensing platform, for on-site and continuous physiological monitoring. We also fabricated wireless, battery-less wearable sensing systems. Utilizing advances in microelectromechanical systems (MEMS) and printing electrode technology, electrochemical sensing devices can now be integrated onto a flexible substrate roughly the size of a coin. These devices are sufficiently small and flexible to be attached to clothing or to the body, to enable long-term pathogen or health status monitoring. We will focus on translating diagnostic protocols to ready-to-use device and instrumentation through three novel techniques. First, we will design and fabricate disposable and flexible sensors using screen printing techniques and printed electronic technology. The PDMS-based stretchable AgNW electrode will be constructed using a bottom-up, fully printed electronic process with a flexible and stretchable PDMS substrate. The stretchable serpentine electrode will be prepared by printed electronic technology and flexible printed circuit board (FPCB) technology. After the construction of the electrode array, specific biomedical modification will be performed to achieve multi-channel biosensing detection of different target substances.

Second, we will design and fabricate portable/wearable electrochemical sensing circuits, including potentiostat modules and microcontroller modules. The potentiostat module directly connects to electrochemical sensing electrodes. The potentiostat generates specific electrical excitations and receives the corresponding voltage/current signals from the sensor. The signals will be further processed by circuits, including low-pass filters, transimpedance amplifiers, and analog to digital converters.

Third, we will implement novel approaches for supplying power and performing data transmission for wireless wearable biosensors. By integrating an NFC module into the sensing device, information can be exchanged with the users’ smartphone, while the phone acts as a conduit to cloud-based service systems to control, analyze, and share information. The collected data can be analyzed locally on the smartphone, or processed by sophisticated machine learning algorithms running on the cloud server.

Pathogen sensing using the capabilities of image sensors in mobile devices

We seek to develop a much more rapid, cost-effective, PCR-free, ultrasensitive COVID-19 diagnosis in point-of-care (POC) settings, in which intact SARS-CoV-2 virions are detected through recognition of distinct viral epitope (antigen) features, in which the resulting fluorescent signal is read using a smartphone-based reader. A high degree of binding avidity and selectivity for the SARS-CoV-2 virus is built on our recent success in understanding the structural aspects of viral surface proteins that can be matched at nanoscale precision by engineered designer DNA nanostructure (DDN) platforms. As discussed in Aim 3, our team recently designed and synthesized a star-shaped DNA architecture to display 10 dengue virus (DENV) envelope protein-targeting aptamers in a 2D-pattern precisely mirroring the complex spatial arrangement of DENV epitopes27. For a COVID-19 sensor, the specific recognition of SARS-CoV-2 can be achieved through a customized DNA nanostructure-based viral probe, which takes the form of repeated rhombus shapes that precisely match the intra- and inter-spatial pattern of SARS-CoV-2 trimeric spike (S) glycoprotein clusters, and integrates a rhombus-shaped array of aptamers that are designed to form maximum affinity and specificity binding interactions with spikes in a polyvalent, pattern-matching fashion.

In the absence of SARS-CoV-2, each fluorophore-tagged aptamer has the fluorescence quenched by a quencher carried by the “aptamer-lock” DNA that forms duplex with the aptamer. When exposed to a patient sample, such as saliva or nasal swab material in solution, the DNA rhombus-shaped “virus nets” rapidly and selectively bind intact virions with maximum avidity through a much stronger aptamer-spike interaction, resulting in the departure of aptamer-lock DNA from the aptamer and in turn the separation between the fluorophore and quencher. The released fluorescent signal is readily detected by our smartphone-based fluorescent reader. We will develop and demonstrate a rapid, room temperature, singlestep, virus-specific, and ultrasensitive assay for COVID-19 that can be performed immediately after sample collection at the point of care, and provide an early detection result in <2 minutes. Using the data generated from this support, we will identify industrial partners for deploying our COVID-19 sensor. As suggested by our DENV sensor study our COVID-19 sensor has promising potential to help monitor and curb the spread of COVID-1977-79, as the best defense against highly contagious infectious diseases is still early detection.

To achieve this goal, we will design and characterize DDNs for high affinity and specificity binding with intact SARS-CoV-2 virions, using SPR assays to measure binding avidity between each virus net and SARS-2. Next, we will demonstrate SARS-2 virus sensing and detection signal reading by our smartphone fluorimeter. To characterize the system performance, we will use the smartphone fluorimeter with virus spiked samples to establish integrated positive/negative experimental controls, the sensing protocol, and determine the limit of detection and linearity, followed by detect SARS-2 from clinical specimens.