Detection of Prostate Specific Membrane Antigen at Picomolar

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Letter

Detection of Prostate Specific Membrane Antigen at Picomolar Levels Using Biocatalysis Coupled to Assisted Ion Transfer Voltammetry at a Liquid-Organogel Microinterface Array Rashida Akter, and Damien W.M. Arrigan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03518 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 9, 2016

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Analytical Chemistry

Detection of Prostate Specific Membrane Antigen at Picomolar Levels Using Biocatalysis Coupled to Assisted Ion Transfer Voltammetry at a Liquid-Organogel Microinterface Array Rashida Akter and Damien W. M. Arrigan* Nanochemistry Research Institute & Department of Chemistry, Curtin University, G.P.O Box U1987, Perth, Western Australia 6845 (Australia) * Email [email protected]; telephone +61-(0)8-9266-9735; fax +61-(0)8-9266-2300 ABSTRACT: A label-free electrochemical strategy for the detection of a cancer biomarker, prostate specific membrane antigen (PSMA), at picomolar concentrations without the use of antibodies, was investigated. The approach is based on the assisted ion transfer of protons, generated by a series of enzymatic reactions, at an array of microinterfaces between two immiscible electrolyte solutions (µ-ITIES). This non-redox electrochemical approach based on biocatalysis-coupled proton transfer at the µITIES array opens a new way to detect the prostate cancer biomarker, with detection capability achieved at concentrations below those indicative of disease presence. The strategy is expected to contribute to cancer diagnostics, recurrence monitoring, and therapeutic treatment efficacy.

Early detection of protein biomarkers is a vital aspect of clinical and biomedical research for disease treatment.1 For instance, early detection of a cancer at stage 1 is associated with a >90% five-year survival rate.2 Of the many types of cancers, prostate cancer (PC) is the most common cancer in men and, after lung cancer, is amongst the leading causes of cancer deaths.3 Generally, prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) biomarkers are considered two important biomarkers in clinical diagnosis of PC. However, PSA is not a specific biomarker4 and PSMA levels in serum are more useful for the reliable indication of PC.5 The normal level of PSMA in serum is ca. 0.25 nM (21 ng/mL), which increases to ca. 3.5 nM (294 ng/mL) in the biological fluids of a PC patient.6 Since serum samples are generally diluted 10-100 times prior to analysis, the development of simple and inexpensive ultrasensitive detection strategies for PSMA with limits of detection (LOD) in the picomolar (pM) region is of utmost importance for the early detection of PC. Conventional detection methods for cancer biomarkers, including PSMA, are typically based on various forms of immunoassyas7-10 such as fluorescence immunoassay (FIA), chemiluminescence immunoassay (CIA), and, most frequently, enzyme-linked immunosorbent assay (ELISA). Although these conventional immunoassays are routinely used in hospitals and clinics, they are costly, complicated to perform, time-consuming, labour-intensive, and less sensitive (~ ng/mL).6,11 Thus, it is crucial to develop an alternative PSMA detection method which should be simple, low-cost, fast, and highly sensitive. In this letter, we demonstrate that PSMA detection can be simply and sensitive-

ly achieved via assisted proton transfer12,13 by a proton selective ionophore, octadecyl isonicotinate (ETH 1778), at an array of microscale interfaces between two immiscible electrolyte solutions (µ-ITIES),14-18 in which the transferred protons are generated through sequential proteolysis by PSMA and enzymatic catalysis of the proteolytic product (Scheme 1). The ITIES19-23 has the advantage that the electrochemical behaviors of species which are not readily oxidized or reduced can be investigated. For example, the electrochemical behaviors of proteins, peptides, and metabolites have been successfully studied in a non-redox manner at the ITIES and µ-ITIES.24-29 Initially, the µ-ITIES array was characterized by voltammetry of tetrabutylammonium (TBA+) ion transfer (Figures S1) and assisted proton (H+) transfer by ETH 1778 (Figure S2). For the TBA+ ion transfer, steady-state and peak-shaped curves on the forward and reverse scans, respectively, were observed which were due to radial and linear diffusion of TBA+ ion in the aqueous and organogel phases, respectively.16 The transfer of TBA+ was reversible and controlled by the diffusion of TBA+ (see supporting information for details). In the case of assisted H+ transfer under either excess H+ (Figure S1B) or excess ionophore (Figure S3A), peak-shaped curves for both forward and reverse scans were observed. Although the peak-shaped curves observed under H+excess conditions are due to the linear diffusion of ionophore and H+-ionophore complex within the organogel phase,16 the peakshaped curves observed under ionophore-excess conditions were unexpected. In this case, radial diffusion of H+ in the aqueous phase was expected to be dominant, producing a steady-state voltammogram. In contrast, a peak-shaped voltammogram was observed during the forward scan. We attribute the peak-shaped voltammogram to the linear flux of the ionophore within the

Scheme 1. Schematic illustration of the PSMA detection principle.

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Figure 1. (A) CVs for PSMA detection via the assisted transfer of H+-generated from the proteolytic reaction of PSMA followed by an enzymatic reaction at various PSMA concentrations: (i) 0, (ii) 10, (iii) 20, (iv) 50, (v) 70, and (vi) 100 ng/mL. The concentrations of NAAG, GDH, NAD+ and ETH 1778 were 2.0 mM, 2.0 mg/mL, 2.0 mM and 10 mM, respectively. The scan rate was 5.0 mV/s. (B) plot of proton transfer current versus PSMA concentration. silicon microchannels supporting the organogel phase.30 At conventional ITIES, in the case of excess ionophore, the observed limiting current should be proportional to the concentration of transferring ion. However, for the water/organogel system, current is limited by the diffusion of the ionophore in the organogel phase even for a concentration ratio of transferring ion:ionophore down to 1:500, because of the much lower diffusion coefficient of ionophore in the organogel phase compared to that of the ions in the liquid aqueous phase.31 The diffusion coefficient of ETH 1778 in the organogel phase was estimated to be 6×10-7 cm2/s, similar to that of a ligand in a PVC-NPOE gel.14 In the present case, the rapid diffusion of H+ in the aqueous phase is not the limiting factor even at the lower concentrations of H+ because of its much higher diffusion coefficient (9.31 ×10-5 cm2/s)32 in the aqueous phase relative to the ionophore in the organogel phase (diffusion coefficient ratio of H+ and ionophore is 1:155). Moreover, the peak-to-peak separation was found to be greater than 60 mV for the assisted H+ transfer, suggesting contributions from some kinetic factors to the peak-shaped curves. Following the successful detection of H+, the detection of the protein biomarker PSMA was evaluated. Scheme 1 illustrates the detection principle examined here, which employs two biocatalytic reactions for generation of protons followed by assisted proton transfer as the final step. In the first biocatalysis reaction, the proteolytic activity of PSMA catalyzes the hydrolysis of N-acetylaspartyl-glutamate (NAAG). In the second reaction, the generated glutamate (Glu) is enzymatically transformed to α-ketoglutarate, NADH, NH4+, and H+ by glutamate dehydrogenase (GDH) in the presence of its cofactor, nicotinamide adenine dinucleotide (NAD+). Finally, the enzymatically generated H+ undergoes assisted ion transfer across the µ-ITIES array by the ionophore ETH 1778 contained in the organogel phase. Figure 1A shows the CV responses obtained in the absence (i) and presence (ii-vi) of various aqueous phase concentrations of PSMA. In the absence of PSMA, no voltammetric peak was observed, whereas in the presence of PSMA, well-resolved peak-shaped curves were observed at the various concentrations of PSMA examined. The peak potentials of these peak-shaped curves were in agreement with those obtained for the ionophore-assisted H+ transfer in the absence of the biochemical reactants (Figure S3 (A)), confirming the potentiality of the proposed concept for PSMA detection. The peak currents increased with PSMA concentration. Thus, a quantitative estimation of PSMA concentration could be evaluated from the

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proposed detection strategy. Figure 1B shows the resulting current versus concentration plot. As expected, the current response was linearly proportional to the concentration of PSMA, yielding a regression equation of I (nA) = (0.189 ±0.047) (nA) + (0.024 ± 7.8 × 10-4) (nA/ng/mL) C (ng/mL), with a regression coefficient of 0.998. The limit of detection (LOD) for PSMA was determined to be 5.9 ng/mL (or 69 pM), based on three times the standard deviation of the blank (n = 3) divided by the slope of the calibration plot. The non-zero intercept in the calibration plot might be due to the capacitance and uncompensated resistance of the cell, which can be minimized with further optimization. Although using CV in this way provides a clinically-relevant linear dynamic range, 10-100 ng/mL (0.117–1.17 nM) and LOD (69 pM), further improvement of the LOD would be beneficial for practical applications in real samples. Before attempting to improve the sensitivity of the PSMA detection by other more sensitive electrochemical methods, the present µ-ITIES array strategy was evaluated for selectivity. To do this, CV responses were recorded for mixtures of PSMA with other common metabolites/proteins such as ascorbic acid (AA), cytochrome c (Cyt c), lysozyme (Lys) and bovine serum albumin (BSA) (Figure 2A). The PSMA concentration was 80 ng/mL (0.95 nM) whereas the AA, Cyt c, Lys, and BSA concentrations were 100, 10, 10, 10, and 10 µM, respectively. It should be noted that AA, Cyt c, and Lys are selected here as model interferences that might be commonly present in biological fluids and potentially interfere in biomolecule detection. As shown in Figure 2A, the peak currents observed for the mixtures of PSMA with the additional species present (curves iii-vi) did not change from that observed for PSMA alone (curve ii) despite the concentrations of the added substances being much higher than that of PSMA. This insensitivity to the added substances was due to the fact that AA, Cyt c, and Lys cannot cleave the NAAG peptide, so that no glutamate, and hence no H+, was produced. Consequently, no assisted H+ transfers occurred at the µ-ITIES array and no significant decrease in current responses were observed even when their concentrations were 5-6 orders of magnitude higher than the PSMA concentration in the mixture. Additional studies will be required to examine whether other proteolytic enzymes that might be present in patient samples could interfere with PSMA detection; however, PSMA, as known as glutamate carboxypeptidase II, acts selectively to cleave C-terminal glutamate residues in NAAG.33 Hence the natural selectivity of PSMA for NAAG will be evaluated in future work to detect PSMA in the presence of other

Figure 2. (A) CVs assessment of selectivity: (i) AA + Cyt c + Lys + BSA, (ii) PSMA, (iii) PSMA + AA, (iv) PSMA + Cyt c, (v) PSMA + Lys, (vi) PSMA + BSA. In all cases, PSMA, AA, Cyt c, Lys, and BSA concentrations were 0.95 nM, 100 µM, 10 µM, 10 µM, and 10 µM, respectively. Other conditions were same as in Figure 1. (B) Bar graph representation of the selectivity performances.

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proteolytic enzymes and in patient sample media. Following selectivity assessment, the detection performance of the proposed strategy was further enhanced by the use of square wave voltammetry (SWV). Figure 3A shows a series of SWVs for various concentrations of PSMA, from 1.0 to 10 ng/mL (0.0117-0.117 nM). The peak currents were directly proportional to PSMA concentration (Figure 3B), yielding a linear regression equation of I (nA) = (0.94 ± 0.025) (nA) + (0.25 ± 0.004) (nA/ng/mL) C (ng/mL) with a regression coefficient of 0.999. The reproducibility, expressed as the relative standard deviation (RSD), was 5.0% (current response: 1.4 ± 0.07 nA) and 5.9 % (3.4 ± 0.2 nA) at concentrations of 2 and 10 ng/mL, respectively. The LOD was determined to be 0.3 ng/mL (3.5 pM), which is ca. 20 times lower than that determined by CV, indicating that SWV may be more useful in the practical detection of PSMA. In comparison to published LODs for label-free PSMA detection, the present LOD was lower than the reported values of 3.1 and 0.01 nM in phosphatebuffered fluoride (PBF) solution and in synthetic urine, respectively,34 56 nM in high ionic strength PBF,35 11.76 nM (10 ng/ml) in phosphate buffered saline (PBS) solution,36 and 9.41 pM (0.8 ng/mL) in buffered solution.37 We note, however, that the LOD of the proposed antibody-free and label-free method is currently inferior to that of automated microfluidic multiplexed nanomaterial-labeled immunoassays (0.1 pg/mL~10 fg/mL).38,39 It is also expected that the linear dynamic range and the LOD could be further improved by optimizing the reaction time and the concentrations of NAAG, GDH, and NAD+. Importantly, the validity of the proposed µ-ITIES detection strategy for PSMA in biological fluids, such as blood serum, urine, etc., needs to be evaluated before attempting practical clinical application in PC patient samples. Although the biosensing of urea, creatinine and glucose using µ-ITIES has been previously reported through assisted NH4+ and H+ ion transfers,40-43 this is the first report of an assisted H+ transfer-based cancer biomarker protein detection through the use of µ-ITIES arrays coupled with proteolytic and enzymatic reaction.

detecting PSMA in biological samples and then applied to PC patient samples. Detection of PSMA without expensive labels and antibodies makes the proposed method simpler and less expensive, thus supporting its eventual use in a wide range of locations to aid in PC diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website. Experimental details including reagents, apparatus, and PSMA detection method, and the characterization of the micro-ITIES array.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Phone +61-(0)8-9266-9735; fax +61-(0)8-9266-2300.

Author Contributions The manuscript has written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT RA thanks the Australian Government for the award of an Endeavour Fellowship (4651-2015) to support her research visit at Curtin University. The microporous silicon array membranes were a gift from Tyndall National Institute, Cork, Ireland.

REFERENCES

Figure 3. (A) SWV detection of PSMA at various concentrations: (i) 0, (ii) 1.0, (iii) 2.0, (iv) 5.0, (v) 7.0, and (vi) 10 ng/mL. The concentrations of NAAG, GDH, NAD+, and ETH 1778 were same as Figure 1. (B) Calibration plot for PSMA detection. In conclusion, a sensitive non-redox electrochemical detection strategy based on assisted H+ transfer voltammetry at a µ-ITIES array is capable of specifically detecting low pM concentrations of the cancer protein biomarker, PSMA, without labelling and without use of antibodies. In the future, the practical applicability of the proposed µ-ITIES detection strategy will be verified by

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