Nanoparticle-based bio-barcode assay redefines "undetectable" PSA and biochemical recurrence after radical prostatectomy

Abstract

We report the development of a previously undescribed gold nanoparticle bio-barcode assay probe for the detection of prostate specific antigen (PSA) at 330 fg/mL, automation of the assay, and the results of a clinical pilot study designed to assess the ability of the assay to detect PSA in the serum of 18 men who have undergone radical prostatectomy for prostate cancer. Due to a lack of sensitivity, available PSA immunoassays are often not capable of detecting PSA in the serum of men after radical prostatectomy. This new bio-barcode PSA assay is approximately 300 times more sensitive than commercial immunoassays. Significantly, with the barcode assay, every patient in this cohort had a measurable serum PSA level after radical prostatectomy. Patients were separated into categories based on PSA levels as a function of time. One group of patients showed low levels of PSA with no significant increase with time and did not recur. Others showed, at some point postprostatectomy, rising PSA levels. The majority recurred. Therefore, this new ultrasensitive assay points to significant possible outcomes: (i) The ability to tell patients, who have undetectable PSA levels with conventional assays, but detectable and nonrising levels with the barcode assay, that their cancer will not recur. (ii) The ability to assign recurrence earlier because of the ability to measure increasing levels of PSA before conventional tools can make such assignments. (iii) The ability to use PSA levels that are not detectable with conventional assays to follow the response of patients to adjuvant or salvage therapies.

Scheme 1.

Schematic representation of the PSA Au-NP probes (Upper) and the PSA bio-barcode assay (Lower). (Upper) Barcode DNA-functionalized Au-NPs (30 nm) are conjugated to PSA-specific antibodies through barcode terminal tosyl (Ts) modification to generate the coloaded PSA Au-NP probes. In a second step, the PSA Au-NP probes are passivated with BSA. (Lower) The bio-barcode assay is a sandwich immunoassay. First, MMPs surface-functionalized with monoclonal antibodies to PSA are mixed with the PSA target protein. The MMP-PSA hybrid structures are washed free of excess serum components and resuspended in buffer. Next PSA Au-NP probes are added to sandwich the MMP-bound PSA. Again after magnetic separation and wash steps, the PSA-specific DNA barcodes are released into solution and detected using the scanometric assay, which takes advantage of Au-NP catalyzed silver enhancement. Approximately ½ of the barcode DNA sequence (green) is complementary to the “universal” scanometric Au-NP probe DNA, and the other ½ (purple) is complementary to a chip-surface immobilized DNA sequence that is responsible for sorting and binding barcodes complementary to the PSA barcode sequence.

Fig. 1.

Representative PSA calibration curve. The calibrator samples were prepared by spiking 0.0, 0.1, 1.0, 5.0, and 10.0 pg/mL of WHO 90:10 PSA standard into human female serum prescreened for PSA (SI Materials and Methods) (Fig. S2). Assuming 100% serum, the final PSA concentrations correspond to a 0.33, 3.3, 16.6, and 33.3 pg/mL calibrator series, accordingly. Samples were run on the automated system with subsequent scanometric detection of the barcode DNA strands released from the 30 nm Au NP probes for PSA target detection and quantification. An example of the gray scale scanometric readout results is shown. The top row of spots in each 2 × 6 well set is the assay response to PSA barcode DNA obtained from the immunoassay, and the bottom set of six spots is the scanometric assay calibrator response. The zero calibrator (no PSA added) is on the far left, with increasing PSA concentrations, as above, moving to the right. The scanometric DNA calibrator sequence is added at the same concentration in each well set.

Fig. 2.

Nonrecurrent patients 1–9. Postprostatectomy PSA concentrations (pg/mL) as measured with the bio-barcode assay (blue squares). ELISA limit of detection (0.1 ng/mL) is outside the scale of the graph as indicated by the red arrow. Xs indicate samples that are undetectable with the bio-barcode assay.

Fig. 3.

Recurrent patients 10–15, who had undetectable PSA values on the first postoperative sample when measured with conventional assays (the commercial immunoassays used to determine the postprostatectomy PSA serum values in the patient cohort changed during the period of patient follow-up. The Abbott IMx assay was used until 2001 and the Bayer Centaur assay was used thereafter. In each case, the clinical limit of PSA detection was 0.1 ng/mL). Postprostatectomy PSA concentrations (pg/mL) as measured with the bio-barcode assay (blue squares) and the commercial assays (red dots) (see above). The clinical limit of detection (0.1 ng/mL) is indicated by a red dotted line, or red arrow when outside the scale of the graph. Xs indicate samples that are undetectable with the bio-barcode assay. Blue asterisks indicate that the PSA value was above the optimal detection range for the bio-barcode assay; therefore, the ELISA value was used.

Fig. 4.

Recurrent patients 16–18, who demonstrated persistent PSA elevations after surgery. Postprostatectomy PSA concentrations (pg/mL) measured with the bio-barcode assay (blue squares) and the commercial assays (red dots). Clinical limit of detection (0.1 ng/mL) is indicated by a red dotted line, or red arrow when outside the scale of the graph. Xs indicate samples that are undetectable with the bio-barcode assay. Blue asterisks indicate that the PSA value was above the optimal detection range for the bio-barcode assay; therefore, the ELISA value was used.