Homogeneous Dual Fluorescence Count of CD4 in Clinical HIV-Positive Samples via Parallel Catalytic Hairpin Assembly and Multiple Recognitions

Regularly measuring the level of CD4+ cells is necessaryfor monitoringprogression and predicting prognosis in patients suffering from aninfection with the human immunodeficiency virus (HIV). However, thecurrent flow cytometry standard detection method is expensive andcomplicated. A parallel catalytic hairpin assembly (CHA)-assistedfluorescent aptasensor is reported for homogeneous CD4 count by targetingthe CD4 protein expressed on the membrane of CD4+ cells. Detectionwas achieved using CdTe quantum dots (QDs) and methylene blue (MB)as signal reporters. CdTe QDs distinguished CHA-assisted release ofAg(+) and C-Ag+-C and MB that hasdifferentiated cytosine (C)-rich single-stranded DNA (ssDNA) and C-Ag+-C, generating changes in fluorescence intensity. Withthe assistance of the CHA strategy and luminescent nanomaterials,this method reached limits of detection of 0.03 fg/mL for the CD4protein and 0.3 cells/mL for CD4+ cells with linear ranges of 0.1to 100 fg/mL and 1 to 1000 cells/mL, respectively. The method wasvalidated in 50 clinical whole blood samples consisting of 30 HIV-positivepatients, 10 healthy volunteers, and 10 patients with cancer or otherchronic infections. The findings from this method were in good agreementwith the data from clinical flow cytometry. Due to its sensitivity,affordability, and ease of operation, the current method has demonstratedgreat potential for routine CD4 counts for the management of HIV,especially in communities and remote areas.