A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer

Sensors 2015, 15, 11972-11987; doi:10.3390/s150511972 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Article A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer Sang Wook Lee 1,*, Kazuo Hosokawa 1, Soyoun Kim 2,*, Ok Chan Jeong 3, Hans Lilja 4,5,6,7, Thomas Laurell 2,8 and Mizuo Maeda 1 1 Bioengineering Laboratory, RIKEN, Saitama 3510198, Japan; E-Mails: k-hos[email protected] (K.H.); [email protected] (M.M.) 2 Department of Biomedical Engineering, Dongguk University, Seoul 100715, Korea; E-Mail: thom[email protected] 3 Department of Mechanical Engineering, Inje University, Gimhae-si 621-749, Korea; E-Mail: mem[email protected] 4 Department of Translational Medicine, Lund University, Skåne University Hospital in Malmö, Malmö 20502, Sweden; E-Mail: [email protected] 5 Departments of Laboratory Medicine, Surgery (Urology), and Medicine (GU Oncology), Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA 6 Institute for Biosciences and Medical Technology, University of Tampere, Tampere 33800, Finland 7 Nuffield Department of Surgical Sciences, University of Oxford, Oxford OX3 7DQ, UK 8 Department Biomedical Engineering, Lund University, Lund 22100, Sweden *Authors to whom correspondence should be addressed; E-Mails: [email protected] (S.W.L); [email protected] (S.K.); Tel.: +81-3-5841-4329 (S.W.L); +82-3-1961-5190 (S.K). Academic Editor: Alexander Star Received: 19 February 2015 / Accepted: 14 May 2015 / Published: 22 May 2015 Abstract: Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macroand nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. OPEN ACCESS Sensors 2015, 15 11973 Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10−4 to 102 ng/mL). Keywords: porous silicon; human kallikrein 2; antibody microarrays; prostate cancer 1. Introduction Human tissue kallikrens (hKs) are secreted from human proteases with diverse expression and physiological roles [1]. They consist of 15 (hK1-hK15) genes located on chromosome 19, in the q13.3–13.4 region. Among them, three kallekreins (hK1, hK2 and hK3) have high amino acid homology each other. hK2 and hK3 (PSA) share 80% amino acids while KLK1 shows 62% to 67% homology with PSA and hK2, respectively [2]. As a biomarker of prostate cancer, the levels of prostate specific antigen (PSA or hK3) in the serum, originating from leakage of pathological tissue to the vascular system, are routinely measured, and have been shown to be proportional to the tumor burden [3]. Even though it is the most commonly used biomarker for diagnosis of prostate cancer, and PSA screening tests are reported to have a significant correlation with 20% reduction in cancer mortality, a PSA concentration above the frequently used diagnosis cutoff (3–4 ng/mL) does not necessarily mean cancer as the serum PSA assay lacks specificity, i.e., the capability of distinguishing prostate cancer from some non-malignant prostatic pathologies such as benign prostatic hyperplasia or prostatitis [4], with the drawback of requiring unnecessary painful biopsies because of the low specificity [5]. It should also be noted that only 25% of men with slightly elevated PSA levels do have PCa [5]. In addition, a PSA concentration below the cutoff may actually be cancer. Moreover there are strong arguments challenging the usefulness of PSA screening tests because of the large discrepancies between decreasing disease aggressiveness and increasing levels of treatment. This has led to widespread criticism that prostate cancer is now an “overdiagnosed” and “overtreated” cancer [6]. Therefore, requirement for additional prostatic tumor markers are increasingly high. Some other kallikrein family proteins are recognized as useful biomarkers of prostate cancer. It is reported that higher KLK4 mRNA levels in the prostate tissue obtained by biopsy are correlated with prognosis and cancer stage [7]. Kallikrein-related peptidase 5 (KLK5) is overexpressed in normal tissues compared to cancerous prostatic ones and it shows an inverse relationship between KLK5 levels and pathologic tumor stage [8]. Moreover, elevated KLK 11 mRNA expressions have been found to be associated with a less advanced stage, and an optimistic disease course for prostate cancer [7]. Human Sensors 2015, 15 11974 kallikrein 2 (hK2) is a serine protease with trypsin-like specificity and has many similarities to PSA. Since the expression of hK2 protein is higher in malignant prostatic tissue versus benign tissue it has been also considered as a prostate cancer (PCa) biomarker since levels of hK2 in serum from PCa patients are increased relative to individuals with BPH [9–12]. Expression analysis by RT-PCR has shown the down-regulation of PSA mRNA, while hK2 mRNA is up-regulated in aggressive tumors [13]. It is suggested that hK2 could also be useful in predicting pathologic stage and grade along with biochemical outcome in patients treated with radical prostatectomy [14]. In patients with mildly elevated PSA levels, hK2 acts as an independent predictor for PCa diagnosis [15]. Moreover, hK2 might be suggested as a potential biomarker in diagnosing poorly differentiated tumors [16], as well as differentiating between organ-confined cancer and extra-capsular disease [17]. The concentration of hK2 in human prostates is approximately 10%–50% of the PSA level and it is 50to 100-fold lower than the PSA concentration in blood serum [18,19]. Despite the intrinsically low expression levels of hK2, development of sensitive assay methods of hK2 is insufficient compared to that of PSA assay [20–24]. Only the Dissociation-Enhanced Lanthanide Fluorescent Immunoassay, the so called DELFIA system that utilizes the unique chemical properties of lanthanide chelates in concert with time-resolved fluorescence (TRF) detection, has reported a limit of detection of hK2 in the low pg/mL with a 103 order (3 pg/mL to 3 ng/mL) dynamic range [10,25]. This method requires laborious labeling procedures and a sophisticated optical system. Therefore, a robust, simple but highly sensitive assay technology for hK2 detection is still required. The 3-D macro-pore structures of the P-Si enlarges the surface area for immobilization [26,27] and hence results in an increased density of capture antibody on the surface [28]. Since physical adsorption is the main method to bind the antibody on the surface, it provides fast immobilization of capture antibody (after less than a few minutes of incubation) without any chemical treatment on the surface [28]. Antibody or protein (such as IgG and Protein A) is strongly adsorbed on the silicon surface when the molecules are dispensed as small droplets and the droplets are quickly dried out [26–28]. Numerous factors, including pore size, protein size, surface chemistry and layer thickness, will influence the amount of protein adsorbed, as well as its structure and function [29,30]. There are hundreds of different cross-linking reagents available, resulting in covalent binding between the biomolecules and silicon surface, however, it is very beneficial to find a surface that adsorbs protein spontaneously since derivation might affect the other surface properties, such as hydrophobicity, fluorescent background and surface charges [30]. P-Si enables one to set up a sensitive and simple assay protocol compared to the other proposed amplification methods such as modifying the detection antibodies, by e.g., dendritic amplification [31], catalyzed signal amplification with colorimetric readout [32,33] or detection with rolling-circle amplification [34]. We previously developed a P-Si (porous silicon) antibody microarray platform for analyzing prostate specific antigen (PSA) in serum [24] and α-synuclein in cerebrospinal fluid (CSF) [35] with high sensitivity and reproducibility. P-Si with sub-micron pores is optimal for antibody immobilization. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA––prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The microarray showed a LOD of 800 fg/mL and a dynamic range of 800 fg/mL to 80 ng/mL in serum-spiked PSA [24]. Usage of our P-Si microarray also extends to α-synuclein in CSF which is one of the potential biomarker of Parkinson’s disease. The Sensors 2015, 15 11975 porous silicon microarray displayed a 35 pg/mL LOD and a dynamic range of four orders of magnitude (17 pg/mL to 500 ng/mL) [35]. The substrate is well suited for surface-based immunoassays because the microand nonporous structure of the substrate adsorbs antibodies in an intact state on the enlarged 3-D surface. It can be noted that this surface is also compatible with mass spectrometric readout [28,36]. Table 1 present several supports materials for microarrays and their respective advantages and disadvantages. Table 1. Solid microarray supports. Derivatized Glass Filter/Membrane Gel Pad/Agarose Film Porous Silicon Advantages Cheap Low cost High sample capacity High sample capacity Compatible with most micro-arrayers Reusable Spot homogeneity Low unspecific binding Disadvantages Non-uniform spots (Coffee ring effect) Limit of spot density Expensive Laborious procedures Laborious procedures The protein/antibody microarray format should ultimately be used not only for qualitative analysis such as profiling the abundance of thousands of proteins [37,38], or globally analyzing protein phosphorylation [39], but also to evolve into the development of a quantitative approach [40,41]. Although the potential of protein microarray technology was foreseen more than 20 years ago by Ekins [42], it is only recently that it started to find its way into proteomics research, diagnosis and drug discovery [43–45]. Sandwich immunoassays are widely used for diagnostics, frequently in 96 well formats. They consist of a pair of antibodies (capture antibody and detection antibody) that bind to the target biomarkers in a sandwich format. Sandwich assays offer high detection sensitivity resulting from the enrichment of the target proteins by the capture antibody [46,47]. In this study, we developed a P-Si-based antibody microarray platform for improving the detection sensitivity of the hK2 assay. Detection sensitivity and dynamic ranges of the P-Si assay platform were investigated by varying the density of the capture antibody on the spots. Figure 1 shows the procedures of the developed hK2 immunoassay. Three different concentrations of capturing antibody were arrayed on a porous silicon chip and evaluated in the detection hK2 in buffer (PBS). The P-Si antibody microarray format, with optimized capture antibody concentration, was subsequently evaluated in the detection of hK2 spiked into human female serum. We also investigate the cross-reaction between hK2 capture antibodies and PSA antigen since PSA and hK2 antigen have high homology [9,10]. HK2 capture antibodies were dispensed on the P-Si chip and PSA spiked serum samples were incubated on the chip for testing the level of cross-reaction. The developed platform has the advantages of low sample consumption, time efficiency and robustness. It took in total time of around 3 h from dispensing the capture antibody on the surface to the end of the immunoassay. Sensors 2015, 15 11976 Figure 1. Schematic of the P-Si chip immunoassay procedure, starting with the dispensing of hK2 capture antibodies onto the porous silicon surface. The porous silicon chip with physically adsorbed antibodies is placed into an assay well made of polydimethyl-polysiloxane (PDMS), and hK2-containing serum samples are added; the size of the PDMS assay is well suited for each P-Si chip and makes it easy to perform parallel pipetting. Subsequently, detection antibody (polyclonal primary and Alexa 488 labeled secondary antibody) is used for measuring fluorescent signals under a microscope. 2. Experimental Section 2.1. P-Si Fabrication The porous silicon fabrication is governed by various etching parameters such as HF concentration, current density, anodization time, illumination, orientation of crystal, silicon types, doping level, etc. [39]. The porous silicon fabrication procedure in this paper followed optimum antibody immobilization conditions described previously [48]. Briefly, silicon, 6–8 Ω·cm resistivity (boron doped p-type), <100> orientation, was purchased from Addision Engineering (San Jose, CA, USA). The wafer was placed in an electrochemical-etching cell. The electrolyte solution consisted of 3.6% hydrofluoric acid and 90.7% dimethylformamide (Merck, Darmstad, Germany). The silicon was anodized for 70 min with backside illumination. Current density during anodization was 90 mA/m2, after which the silicon was washed in ethanol three times and diced into 3 mm × 3 mm pieces to fit a microtiter plate format (Corning Costar Corporation, Cambridge, MA, USA). Sensors 2015, 15 11977 2.2. PDMS Well A 45 mm× 70 mm size and 5 mm thickness polydimethylpolysiloxane (PDMS) slab was prepared to well format as seen in Figure 2. The slab was fabricated with a Syglad 184 kit (Dow Corning Toray, Tokyo, Japan) that contains a PDMS monomer base and a curing elastomer reagent. The base and curing reagent were mixed in 10:1 weight ratio, and poured over a flat surface (i.e., silicon or glass). The mixture was kept at 0.02 MPa for 30 min in a vacuum chamber to remove the trapped air bubbles from the liquid PDMS. After removing the bubbles, the mixture were baked at 70 °C for 1 h. Finally, 9 mm holes were punched on the PDMS slab to set up PDMS wells. Figure 2. Porous silicon (P-Si) matrix used for microarrays. P-Si chips and PDMS wells (a) Capture antibody spotted P-Si chips were located in the wells to start the immunoassay. The scanning electron micrographs show a sequential zoom into a typical surface; (b) Macro-pores of micrometer size are clearly seen, combined with a microand nano-morphology (pore size around sub-µm to µm). 2.3. Proteins and Reagents The monoclonal mouse antibody against hK2 (6B7, ab40749) was obtained by Abcam (Cambridge, UK). The polyclonal goat anti–hK2 antibody (PAB7226) and Alexa Fluor 488 labeled donkey anti-goat (ab150129) antibody were obtained at Abnova (Taipei City, Taiwan) and Abcam, respectively. Recombinant hK2 (ATGP2175) was obtained from ATGen (Pankyo, Korea) and prostate specific antigen (PSA) from human semen was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.4. Analytical Samples Female single donor serum was purchased from BBI Solutions (S122–1, Cardiff, UK) and stored at −80 °C following the company’s recommendations. The serum was spiked with the recombinant hK2 in a titration series ranging from 100 fg/mL to 100 ng/mL. Sensors 2015, 15 11978 2.5. Sandwich Assay Monoclonal mouse capture antibody (6B7) for hK2 was dispensed onto the porous silicon chip, 3 × 3 mm size, using a piezoelectric dispenser (DW Scienion AB, Berlin, German) with a spot to spot distance of 150 µm. The antibodies were immobilized on the chip surface by physical adsorption. A drop volume was around 300 pL and a 13 × 13 (169 spot) array was spotted on the surface for each array. The microarray chips were placed in the PDMS well plates for the immunoassay. The porous silicon microarray immunoassay was evaluated using samples with hK2 spiked into both pure buffer (PBS) and female serum to obtain a final concentration ranging from 100 fg/mL to a few 100 ng/mL. The following steps were performed at room temperature: 1 After arraying the antibody, the chips were washed three times using 10 mM PBS to remove loosely bound antibodies. 2 The chips were incubated for 1 h in 100 µL blocking solution (5% (w/v) non-fat dry milk in PBS (Bio-Rad, Hercules, CA, USA)) to prevent non-specific binding and washed 3 times using 0.05% Tween 20 in 10 mM PBS. 3 Following blocking, the chips were incubated with 15 µL of spiked sample for 1 h, then washed (as above) and subsequently incubated with 15 µL of detection antibody (polyclonal goat anti-hK2). 4 After another washing step, 15 µL of AF488 labeled anti-goat polyclonal antibody was added onto the chips and incubated for 1 h. 5 Finally, the chips were washed three times and dried at room temperature. 6 The fluorescent spots images were observed using a eclipse TE2000-U fluorescence microscope (Nikon, Tokyo, Japan). 7 The captured images were analyzed by the open source image-processing tool Image J. 2.6. Mean Spots Intensities and Limits of Detection The mean intensities of spots were measured and quantified by Image J among all microarray spot images, we selected nine spots (a 3 × 3 array) on each P-Si chip for quantification of the data since they fitted well in a single screen image. A total of 18 spots were chosen for data analysis since all experiments were performed on two independent chips. The spot intensities (S) were measured and quantified across the area. The local background (B) was collected in the same way and subtracted from the spot signals, generating mean spots intensities (S-B) as presented in the graphs. Mean spot intensity (S-B) can be defined as ∑( − )/  . The limit of detection (LOD) was followed 3-sigma () defined as the lowest detectable signal from 3-sigma standard deviation above the mean spot intensities of negative control (N). The choice of LOD can be written as >(−) +3, when ( − ) is mean spot intensity of negative control (N) and  is standard deviation of negative control. 3. Results and Discussion 3.1. General Remarks Surface bound immunoassays generally become more sensitive when the affinity or/and the density of the capture antibody increases [48]. The 3-D morphology of the micro/nano porous silicon surface Sensors 2015, 15 11979 layer offers a high antibody immobilization capacity and hence an increased antibody density per spot area [24,28]. Figure 2 shows macroporous layers with a characteristic size of sub micro −1 µm on the silicon surface recorded by Field Emission Scanning Electron Microscopy (FESEM). 3.2. hK2 Assay Performance against Density of Capture Antibody To optimize the P-Si hK2 immunoassay, we first evaluated the assay sensitivity by arraying the capture antibody, 6B7, at three different concentrations and performing the hK2 immunoassay in PBS buffer (10 mM, pH 7.2). Figure 3 shows a titration series of hK2 against three different capture antibody concentrations (75, 100 and 145 µg/mL). Figure 3. Titration series of hK2 in buffer (PBS) solution at three different concentrations of the capturing antibody 6B7 (75 µg/mL, 100 µg/mL and 145 µg/mL). The LOD was found to be 1 pg/mL when the capturing antibody was 75 µg/mL and was reduced to 100 fg/mL when the capturing antibody concentrations were 100 µg/mL. The LOD became 1 pg/mL again when concentration of the antibody was 145 µg/mL. Increasing concentrations of capture antibody yielded elevated fluorescent signals. However, the negative control (N) signal also increased at higher density of the antibody. The coefficient of variance (C.V) and mean spot intensities of arrays were shown in a right side of the figure. The fluorescent signal level of negative control (N) increased up to 70% (mean spot intensity is around 3) at a capture antibody concentration of 145 µg/mL compared to those at 75 and 100 µg/mL (mean spot intensity is around 2 at capture antibody 75 µg/mL and 1.95 at 100 µg/mL). In most cases, spot reproducibility within the chips shows good reliability (CVs~10%–20%) and also was in agreement with earlier results [33]. The linear regression of the mean spot intensities versus hK2 concentration corresponded to a coefficient of determination (R2) equal to 0.97, 0.99 and 0.906 for capture antibody concentrations of 75, 100 and 145 µg/mL, respectively. It was hypothesized that the unspecific binding between the capture antibody and the secondary or the detection antibody increased at elevated capture antibody concentrations. To define the limit of detection, we chose 3-sigma (σ) method, which is the lowest signal detection at least three times above Sensors 2015, 15 11980 the standard deviation of the negative control (N). At a capture antibody concentration of 75 µg/mL the limit of detection (LOD) was found to be 1 pg/mL and at a capture antibody concentration of 100 µg/mL the LOD was improved by one order of magnitude to 100 fg/mL. We believe that the 100 fg/mL LOD reported for hK2 is competitive with the results of the DELFIA system for which a sub pg/mL LOD is reported [10,25]. A further increased density of capture antibody (145 µg/mL) did not yield an improved LOD as this also resulted in a significantly increased negative control level. The dynamic range was 105 (10−3 to 102 ng/mL) at a capture antibody concentration of 75 µg/mL and increased to 106 (10−4 to 102 ng/mL) when the capture antibody concentration was 100 µg/mL. The signal intensities were enhanced in proportion to the concentration of the capturing antibody, which followed the expectations of improved LOD with increased surface density of the capture antibody. However in case of 145 µg/mL capture antibody, the readout signal intensity drastically increased up to 100 pg/mL of hK2 level and it was saturated at higher concentrations (over 1 ng/mL hK2 level). The dynamic range therefore, defined a 102 order (from 1 pg/mL to 100 pg/mL), which was reduced by two orders of magnitude. 3.3. hK2 Immunoassay in Female Human Serum We evaluated the P-Si immunoassay platform subsequently in human serum since the final purpose of our platform development is to quantify the levels of hK2 in clinical samples. Initially, a high concentration of recombinant hK2 (over a few µg/mL) was spiked into a serum sample and the spiked samples were serially diluted up to 106 times with serum to achieve total hK2 levels ranging from a hundred fg/mL to a hundred ng/mL. As a negative control we assayed unspiked serum. Figure 4. hK2-spiked human female serum analyzed with the sandwich microarray at two different capturing antibody concentrations (75 µg/mL and 100 µg/mL). The LOD was 10 pg/mL and 1 pg/mL when the concentrations of capture antibody were 75 µg/mL and 100 µg/mL, respectively. Increased assay sensitivity was observed with an elevated concentration of the capturing antibody (6B7). The negative signal also increased at the higher concentrations of the capture antibody. Sensors 2015, 15 11987 45. Sebastiani, V.; Botti, C.; di Tondo, U.; Visca, P.; Pizzuti, L.; Santeusanio, G.; Alo, P.L. Tissue Microarray Analysis of FAS, Bcl-2, Bcl-x, ER, PgR, Hsp60, p53 and Her2-neu in Breast Carcinoma. Anticancer Res. 2006, 26, 2983–2387. 46. Gembitsky, D.S.; Lawlor, K.; Jacovina, A.; Yaneva, M.; Tempst, P. A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation. Mol. Cell. Proteomics 2004, 3, 1102–1118. 47. 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