Biosensors and Bioelectronics br Detection
Biosensors and Bioelectronics 126 (2019) 581–589
2.6. Detection of ApoA1 in normal Tirapazamine for recovery studies
Approximately 50 mL of clean-catch urine (the first morning mid-stream voided urine from healthy donors) conserved with 1 mM sodium azide as an antibacterial agent. The collected samples were immediately centrifuged at 1500 g, at 4 °C for 10 min. The urine was spiked with various concentrations of ApoA1 protein (20, 50, and 100 ng/mL) for recovery tests. In brief, 100 µL of urine solution spiked with ApoA1 at diﬀerent concentrations was added to the tube containing biochipApoA1 and incubated for 30 min at room temperature; the biochipApoA1 was washed with TBST three times, and then incubated with 150 µL of self-linkable PMGOs (PMGO-1 + PMGO-2 + PMGO-3) for another 30 min developing through the addition of 100 µL of H2O2 + TMB solution and then the addition of HCl. The A450 nm was recorded to determine the ApoA1 concentration in urine using the respective calibration curves for the colorimetric immunosensing using the SpectraMax M2. All re-covery measurements were performed in triplicate for accurate calcu-lations to develop a standard protocol.
2.7. Colorimetric immunosensor for BC diagnosis and prognosis monitoring
This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (IRB: 102–3642A3). Approximately 50 mL of clean-catch urine from four healthy donors and four patients with BC were immediately centrifuged at 1500 g, at 4 °C for 10 min. The sample for the immunosensor and ELISA analyses was collected from the supernatant and stored at −80 °C. The seven urine samples from the patients with high-grade BC were collected before standard clinical treatment, three urine samples from the patients with low-grade BC were collected before standard clinical treatment, and six urine samples from the patients with high-grade BC were collected after standard clinical treatment and no recurrence was observed. For actual experi-ment, briefly, 100 µL of a urine sample was added to the tube con-taining biochipApoA1 which is fixed on the tube bottom then incubated for 30 min at room temperature; the biochipApoA1 was washed with TBST three times, and then incubated with excess of self-linkable PMGOs (50 µL PMGO-1 + 50 µL PMGO-2 + 50 µL PMGO-3) for an-other 30 min. The unbound PMGOs were washed out with TBST by shaking the tube, developing through the addition of 100 µL of H2O2
+ TMB solution and then the addition of 10 µL HCl (1 M). The A450 nm was recorded to determine the ApoA1 concentration in urine using the respective calibration curves for the colorimetric immunosensing. with the SpectraMax M2. Furthermore, we used a human ApoA1 ELISA kit (Arigo Biolaboratories Co., Taiwan), and followed the manufacturer's protocols to determine ApoA1 concentrations in human serum samples for comparison with the results from the immunosensor. All recovery measurements were performed in triplicate for accurate calculations to develop a standard protocol.
3. Results and discussion
3.1. Characterization and confirmation of prepared glassy biochips and PMGO
We envisioned a signal amplification approach to obtain a colori-metric immunosensor for determining ApoA1 concentration in patients’ urine. The ultrasensitive immunosensor was constructed with biochipApoA1 and self-linkable peroxidase-mimic PMGO. After the samples were incubated and ApoA1 was captured on the biochipApoA1, the PMGO-1 was functionalized with AbApoA1 and mouse IgG (PMGO-1), and then rabbit anti-mouse IgG antibody (PMGO-2) and goat anti-rabbit IgG antibody (PMGO-3) were added together. We envisioned that each captured ApoA1 protein should allow for the retention of a large amount of PMGO through a self-linking process, thereby oxidizing more H2O2 + TMB for the amplification of absorbance signals, as well as increasing the analytical performance of the immunosensor for ApoA1