Fluorometric Viral miRNA Nanosensor for Diagnosis of Productive (Lytic) Human Cytomegalovirus Infection in Living Cells

Abstract

A human cytomegalovirus (HCMV) causes a persistent asymptomatic infection in healthy individuals and possesses unexpected dangers to newborn babies, immunocompromised people, and organ transplant recipients because of stealth transmission. Thus, an early and accurate diagnosis of HCMV infection is crucial for prevention of unexpected transmission and progression of the severe diseases. The standard method of HCMV diagnosis depends on serology, antigen test, and polymerase chain reaction-based nucleic acid detection, which have advantages for each target molecule. However, the serological test for an antibody is an indirect method assuming the past virus infection, and antigen and viral nucleic acid testing demand laborious, complex multistep procedures for direct virus detection. Herein, we present an alternative simple and facile fluorometric biosensor composed of a graphene oxide nanocolloid and fluorescent peptide nucleic acid (PNA) probe to detect the HCMV infection by simply monitoring the virally encoded microRNA as a new biomarker of lytic virus infection. We verify the sensing of HCMV-derived microRNA accumulated within 72 h after HCMV infection and examine the diagnosis of HCMV in living cells. We proceed with the time course and concentration-dependent investigation of hcmv-miRNA sensing in living cells as a direct method of HCMV detection at the molecular level on the basis of an intracellular hcmv-miRNA expression profile and graphene oxide nanocolloid-based simple diagnostic platform. The fluorometric biosensor enables the sequence-specific binding to the target HCMV miRNAs in HCMV-infected fibroblasts and shows the quantitative detection capability of HCMV infection to be as low as 4.15 x 10(5) immunofluorescence focus unit (IFU)/mL of the virus titer at 48 h post-infection with picomolar sensitivity for HCMV miRNA.