Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

Abstract

Background: Prostate-specific antigen (PSA), a serine protease, is a biomarker for preoperative diagnosis and screening of prostate cancer and monitoring of its posttreatment.

Methods: In this work, we reported a colorimetric method for clinical detection of PSA using gold nanoparticles (AuNPs) as the reporters. The method is based on ascorbic acid (AA)-induced in situ formation of AuNPs and Cu²⁺-catalyzed oxidation of AA. Specifically, HAuCl₄ can be reduced into AuNPs by AA; Cu²⁺ ion can catalyze the oxidation of AA by O₂ to inhibit the formation of AuNPs. In the presence of the PSA-specific peptide (DAHSSKLQLAPP)-modified gold-coated magnetic microbeads (MMBs; denoted as DAHSSKLQLAPP-MMBs), complexation of Cu²⁺ by the MMBs through the DAH-Cu²⁺ interaction depressed the catalyzed oxidation of AA and thus allowed for the formation of red AuNPs. However, once the peptide immobilized on the MMB surface was cleaved by PSA, the DAHSSKLQ segment would be released. The resultant LAPP fragment remaining on the MMB surface could not sequestrate Cu²⁺ to depress its catalytic activity toward AA oxidation. Consequently, no or less AuNPs were generated.

Results: The linear range for PSA detection was found to be 0~0.8 ng/mL with a detection limit of 0.02 ng/mL. Because of the separation of cleavage step and measurement step, the interference of matrix components in biological samples was avoided.

Conclusion: The high extinction coefficient of AuNPs facilitates the colorimetric analysis of PSA in serum samples. This work is helpful for designing of other protease biosensors by matching specific peptide substrates.

Keywords: Cu2+ ion; ascorbic acid; colorimetric assay; gold nanoparticles; prostate-specific antigen.

Figure 1

(A) Cleavage of peptide DAHSSKLQLAPP by PSA and the structure of ATCUN–Cu²⁺ complex formed between DAHSSKLQLAPP and Cu²⁺. (B) SEM image and EDS elemental mapping images of the Au-coated MMBs. (C) Schematic representation of the MMB-based method for PSA detection. Abbreviations: ATCUN, amino terminal copper and nickel; AuNPs, gold nanoparticles; EDS, energy-dispersive spectroscopy; MMBs, magnetic microbeads; PSA, prostate-specific antigen; SEM, scanning electron microscope.

Figure 2

(A) Change of 100 µM AA absorbance as a function of reaction time in the absence and presence of different Cu²⁺ species. The concentrations of peptide and Cu²⁺ are 3 and 2 µM, respectively. The absorbance values plotted have excluded that contributed by the buffer and individual peptide. (B) UV–Vis absorption spectra of 50 µM AA before and after incubation with the mixture of Cu²⁺ (0.5 µM) and peptide-functionalized MMBs. Abbreviations: Abs, absorption; AA, ascorbic acid; MMBs, magnetic microbeads; UV–Vis, ultraviolet–visible.

Figure 3

UV–Vis absorption spectra and photographic images (A) of HAuCl₄ and CTAC after incubation with different solutions. In curve a, AA (250 µM) was not incubated with Cu²⁺ and peptide-functionalized MMBs. In curves/tubes b and c, AA (250 µM) was preincubated with the mixture of 1.5 µM Cu²⁺ and peptide-functionalized MMBs for 30 minutes. TEM images of the AuNPs generated via the reduction of HAuCl₄ by AA that has been incubated without (B) and with (C) Cu²⁺/DAHSSKLQLAPP-MMBs. Abbreviations: Abs, absorption; AA, ascorbic acid; AuNPs, gold nanoparticles; CTAC, hexadecyltrimethylammonium chloride; MMBs, magnetic microbeads; TEM, transmission electron microscope.

Figure 4

Dependence of the absorption intensity of AuNPs suspension at 530 nm upon the used concentration of AA (10, 50, 100, 150, 200, and 250 µM) (A), Cu²⁺ (0.01, 0.1, 0.5, 1, 1.5, and 2 µM) (B), and DAHSSKLQLAPP-functionalized MMBs (0.5, 1, 2, 3, 4, and 5 mg) (C). In panels B and C, the concentration of AA used is 200 µM. The Cu²⁺ concentration in panel C is 1.5 µM. Abbreviations: Abs, absorption; AA, ascorbic acid; AuNPs, gold nanoparticles; MMBs, magnetic microbeads.

Figure 5

(A) UV–Vis absorption spectra and photographic images of the generated AuNPs in the cases that the DAHSSKLQLAPP-functionalized MMBs have been preincubated with different concentrations of PSA (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 2 ng/mL). (B) Dependence of the absorption intensity of the generated AuNPs on PSA concentration. Abbreviations: Abs, absorption; AuNPs, gold nanoparticles; PSA, prostate-specific antigen; MMBs, magnetic microbeads.

Figure 6

(A) Selectivity of the method: PBS blank (bar 1), 10 ng/mL thrombin (bar 2), 10 ng/mL trypsin (bar 3), 50 ng/mL hemoglobin (bar 4), 50 ng/mL BSA (bar 5), 200 µM AA (bar 6), and 0.8 ng/mL PSA (bar 7). (B) Assays of PSA in the serums of healthy donors and prostate patients. Abbreviations: AA, ascorbic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PSA, prostate-specific antigen.