An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus

Abstract

Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research.

Figure 1. Optimization of ECL-detection conditions.

Optimal ECL-detection conditions were determined by using nine different ECL-emitters/coreactant combinations. (A) Integrated ECL-intensity of constant concentration (25 μM) of three different ECL-emitters (RUB, Ru(bpy)₃²⁺, and DPA) with various concentrations of three different co-reactants (BPO, DBAE, and TPrA). (B) ECL-intensity of 25 μM RUB as a function of potential for different co-reactants (concentrations indicated in figure). (C) ECL-response profiles of different concentrations of RUB with 10 mM BPO as co-reactant. A linear relationship between the integrated ECL intensity and the logarithm of RUB concentration is shown in inset. All ECL-intensity measurements were carried out in MeCN solution containing 0.10 M TBAP supporting electrolyte after degassed with N₂ for 7 min at a 2-mm diameter Pt electrode with a scan rate of 100 mV/s. ECL-intensity of each sample was measured for three times. All experiments were performed in duplicates and repeated at least one time.

Figure 2. Assay design and preparation of immuno-conjugated and ECL-loaded beads.

(A) Diagram of ECL-based immunoassay for detection of viruses. (B) Image of rubrene loaded PSBs (golden yellow, right) taken under UV-light (385 nm excitation) in a confocal microscope. (C) Antibody conjugation to PSBs and MBs was analyzed by immunoblotting assay. All experiments were performed in duplicates and repeated at least one time.

Figure 3. Immuno-conjugated and ECL label loaded polystyrene beads capture ZIKV.

(A) Photograph of a microfuge tubes after addition of ZIKV containing samples to ZV2-PSB(RUB), showing the aggregation of PSBs at the bottom of tube in the presence of ZIKV. (B) Photograph of a microfuge tubes after magnetic separation showing the free MBs (right), and binding of PSBs to MBs (left) in the presence of ZIKV. (C) A phase-contrast image of PSB < ZIKV > MB aggregates showing the binding of anti-ZV2-MB (1 μm diameter, arrow) to the surface of anti-ZV2-PSB (10 μm diameter) in the presence of ZIKV. (D) PSB < ZIKV > MB aggregates that were obtained from samples containing different PFUs of ZIKV were analyzed by RT-qPCR assay to quantify copy number of ZIKV envelope gene. All experiments were performed in duplicates and repeated at least one time.

Figure 4. Anti-ZIKV-PSB specifically detects ZIKV in a highly sensitive manner.

Samples containing different amounts of ZIKV (0–10⁴ PFU) were prepared in PBS containing 2% BSA and allowed to form PSB < ZIKV > MB complexes by reacting with ZV2-PSB(RUB) and ZV2-MB. PSB < ZIKV > MB complexes were separated magnetically and subjected to ECL-intensity measurement. (A) ECL-response curves of PSB < ZIKV > MB aggregates obtained from samples containing different PFUs of ZIKV in PBS. (B) ECL-response of free (unliganded) PSB(RUB) that remained in solution after magnetic separation. (C) A calibration curve of mean ECL intensity of magnetically separated PSB < ZIKV > MB aggregates (upward line, black) and free PSB(RUB) (downward line, blue) that remained in solution after magnetic separation. (D) ECL response of PSB < ZIKV > MB aggregates obtained from samples containing related (DENV and WNV) or unrelated (CHIKV) viruses (10³ PFU) prepared in PBS containing 2% BSA. Sample containing no virus was used as a control to determine background signal (~0, see Fig. 4C). All experiments were performed in duplicates and repeated at least one time.

Figure 5. ECL-based immunoassay detects ZIKV in human biofluids.

Samples containing different amounts of ZIKV (0–10³ PFU) were prepared in human urine or plasma specimens collected from healthy human volunteers. ECL-response from PSB < ZIKV > MB aggregates obtained from (A) plasma, and (B) urine samples containing different PFUs of ZIKV. ECL-response of virus-free PSB(RUB) that remained in solution after magnetic separation from (C) urine and (D) plasma samples. A calibration curve of mean ECL intensity of magnetically separated PSB < ZIKV > MB aggregates (upward line, black) and free PSB(RUB)(downward line, blue) that remained in solution after magnetic separation were generated from (E) urine and (F) plasma samples. No virus control was used to determine background signal. All the experiments were performed in duplicates and repeated at least one time.