ABSTRACT
Despite tremendous advances in treatment of HIV/AIDS and the decrease of HIV incidence, the overall infected
population continues to grow. Progress on prevention of HIV transmission remains far too slow. It is estimated
that 20% of new HIV infections are due to transmission from unaware infected individuals. Hence, early detection
of HIV is particularly important for lowering transmission rates. To this end, extending testing accessibility beyond
clinical settings through self-tests is highly desirable. HIV self-testing is a process in which an individual who
wants to know his/her HIV status collects a specimen, performs the test, and interprets the result in private.
Current HIV self-testing technologies include rapid protein tests and nucleic acid tests. The suboptimal sensitivity
of current protein tests can only support antibody detection and will miss a significant portion of acute infections.
Although nucleic acid tests can reach lower detection limit through amplification technologies such as PCR, they
will miss the information that antigens can provide. Most current research efforts on HIV self-testing are focused
on nucleic acid tests. However, there is no evidence showing that HIV RNA appears ahead of antigen. The major
technological challenge for antigen detection is that proteins cannot be amplified like nucleic acids, leading to
the widely held belief that antigen tests are relatively insensitive and therefore have a limited clinical utility.
We previously demonstrated a click chemistry amplified nanopore (CAN) assay method for ultrasensitive antigen
quantification. This assay achieved 0.5 pg/ml detection limit for HIV-1 p24 antigen and demonstrated reliable
detection in clinical samples from patients missed by nucleic acid and/or ELISA assays. Quantitative p24 results
from this method also indicated correlation between p24 and viral load, suggesting potential use for monitoring
antiretroviral therapy adherence to minimize treatment failure. Based on the CAN assay, this project aims to
develop an ultrasensitive quantitative HIV-1 p24 antigen self-test to improve early detection of acute infections
and monitoring treatment efficacy. The test will be based on a streamlined automatic device including a cost-
effective microfluidic chip for sample preparation and a nanopore reader for laypersons to test themselves using
fingerprick blood. The R61 phase will develop a self-testing device for quantification of HIV-1 p24 antigen at = 2
pg/ml in whole blood to support detection of acute infection and treatment failure. The R33 phase will perform a
clinical evaluation at the Prisma Health Immunology Center to assess the performance, usability, and
acceptability of the self-testing device. Through innovations in click chemistry amplified nanopore detection and
microfluidic sample preparation, we anticipate the test would be able to quantify HIV-1 p24 antigen at as low as
0.5 pg/ml directly from 100µl or less finger prick blood and establish correlation between p24 and viral load levels.
If successfully developed and validated, this CAN self-testing device should enable routine HIV self-testing as
simple as a blood sugar test at home to support global HIV diagnostic and therapeutic efforts.