Disperse-and-Collect Approach for the Type-Selective Detection of

Nov 28, 2016 - ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Future Industries Institute, University of South Australia, Maw...
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Disperse-and-Collect Approach for the Type-Selective Detection of Matrix Metalloproteinases in Porous Silicon Resonant Microcavities Fransiska S. H. Krismastuti, Melissa R. Dewi, Beatriz Prieto-Simon, Thomas Nann, and Nicolas H. Voelcker ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00442 • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Disperse-and-Collect Approach for the Type-Selective Detection of Matrix Metalloproteinases in Porous Silicon Resonant Microcavities Fransiska S. H. Krismastuti1,2, Melissa R. Dewi1, Beatriz Prieto-Simon1, Thomas Nann3, Nicolas H. Voelcker1,* 1

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Future Industries Institute, University of South Australia, Mawson Lakes, Adelaide, South Australia 5095, Australia 2 Research Centre for Chemistry, Indonesian Institute of Sciences, PUSPIPTEK, Serpong, Tangerang Selatan, Banten 15314, Indonesia 3 MacDiarmid Institute, School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington 6140, New Zealand Email: [email protected] Keywords: magnetic nanoparticles, multiparametric detection, matrix metalloproteinases, porous silicon resonant microcavity, chronic wound management ABSTRACT: We report on the design and testing of photonic biosensors for the type-selective detection of different types of matrix metalloproteinases (MMPs). The ability to detect a panel of different MMP types has important implication for prognosis of wound healing. We combine the immunocapture of MMPs on dispersed magnetic nanoparticles modified with antibodies specific for target MMPs (immuno-magNPs) with subsequent MMP detection upon fluorogenic peptide cleavage in porous silicon resonant microcavity (pSiRM) architectures. We achieve fast, sensitive and type-selective detection of MMPs directly in wound fluid. This study sets the scene for downstream developments of multiparametric biosensors as point-of-care (POC) prognostic tools that may step-change chronic wound management.

Chronic wounds are increasing in prevalence in ageing societies and have become one of the key health challenges of the 21st century. Diagnostic devices for a panel of wound biomarkers that are as simple to operate as a blood glucose test for diabetes or pregnancy test would reshape the landscape of chronic wound management. Since the understanding of wound fluid constituents that presage wound healing trajectories has progressed dramatically, research and innovation in developing novel diagnostic and prognostic tools for wound management is on the advance.1 Wound sensors in general pursue two conceptually different approaches: dipstick-type sensors for harvested wound fluid and continuous wound monitors embedded in wound dressings.2-4 Two examples highlight the recent development of POC devices for chronic wounds. Worsley et al.5 demonstrated quantitative detection of two biomarkers, interleukin 6 (IL6) and tumour necrosis factor alpha (TNFα), in a single test strip. This assay was based on a nitrocellulose membrane modified with anti- TNFα and IL6 immobilized on fluorescence microspheres (200 nm diameter). The signal detection was via a lateral flow reader equipped with a detector. Using this ‘test strip’ set-up, they were able to quantitatively detect two biomarkers in a single measurement. The second example by Brocklesby et al.2 monitors bacterial infections in wounds via a bandage containing a blue dye anchored to cephalosporin. Bacterial enzymes enzymatically hydrolyze the cephalosporindye bond, releasing the dye into the wound environment and

changing the bandage color, which can be used as an alert of infection for the patients or clinicians. We have previously published a pSiRM-based biosensor for the detection of MMPs, proteolytic enzymes involved in wound healing.6,7 Our previous biosensor employed a fluorogenic MMP peptide substrate combined with the fluorescenceenhancing properties of the pSiRM structure via the Purcell effect8,9 to achieve ultrasensitive MMPs detection within a few minutes. The MMP level detected corresponded to the total level of MMP-1, -2, -3 and -9 (not for one specific type of MMP) since the fluorogenic peptide immobilized on the pSiRM sensing platform served as a substrate for those four types of MMPs.10 MMPs are classified into five groups, namely the collagenases, the gelatinases, the stromelysins, the matrilysins and the membrane-type MMPs.11-13 The expression and concentration of the MMP change according to the phases of wound healing.14-16 For example, high expression of MMP-9 is normally associated with the inflammatory phases when neutrophils and macrophages infiltrate to the wounded area to phagocyte bacteria. In a poorly healing wound, the MMP-9 level is increased.14,17 The level of MMP-9 decreases when the healing progresses into the proliferative phases and this is followed by high MMP-1 expression.14-16,18 Gutiérrez-Fernández et al. observed that the presence of MMP-8 in wound environment is necessary for wound closure and healing.19 Distinguishing between different MMPs is hence important to prognose

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wound healing phases and also healing status and our previous total MMP biosensor falls short of meeting the requirement for type-selective and multiparametric analysis. Here, we show the use of antibody-displaying magnetic nanoparticles to engineer selectivity into the pSiRM-based biosensor towards the detection of different types of MMP. Carboxy-terminated magnetic nanoparticles (magNP) of 10 nm size were modified with MMP antibodies (MMPAb), with affinity for one type of MMP. The MMPAb-modified immuno-magNP were then incubated in buffer containing MMPs or wound fluid sample to harvest the targeted MMP. After binding MMP, the immuno-magNPs were captured to eliminate interferences and then released on a fluorogenic peptidefunctionalized pSiRM. The active MMP, bound on the immuno-magNPs, cleaved the immobilized MMP peptide and the fluorescence from the fluorogenic dye ((5-[(2-aminoethyl)amino]naphthalene-1-sulfonyl, EDANS) was enhanced by the pSiRM (Figure 1). We confirmed that this approach was successful in detecting and distinguishing between MMP-1 and MMP-9 in a simple buffer solution and also human chronic wound fluid.

Figure 1. Schematic of disperse-and-collect mechanism using magNPs and MMP detection in pSiRM. Upon binding of MMPs to magNPs-MMPAb from a complex sample, magNPs are captured on a magnetic column, while other components of the sample pass through the column and are eliminated. Then, magNPs are directly released on the functionalized pSiRM surface. Finally, MMPs bound to magNPs-MMPAb cleave the fluorogenic peptide resulting in fluorescence emission enhanced by the confinement provided by the pSiRM structure.

EXPERIMENTAL SECTION Synthesis of Carboxylic Acid Terminated Magnetic Nanoparticles (magNPs). The magNPs were prepared from 1.9 g iron oleate paste and 0.31 g oleic acid in 10 mL 1-octadiene solution. The mixture was heated to 200 °C and held at this temperature for 1 h. Once the temperature reached 200 °C, the mixture was heated to its refluxing temperature (320 °C) with a constant heating rate of 8 °C/min and maintained at this temperature for 30 min. The magNPs were then cooled down to room temperature, washed by adding ethanol solution (1:5 volume ratio of nanoparticle solution/ethanol) and recovered by centrifugation at 19000g for 10 min. Further washing was necessary to remove excess ligands for two times at 19000g for 10 min. The precipitate was dissolved in toluene.20 The magNPs produced had hydrophobic ligands which were then replaced by oxalic acid leaving the magNPs carboxylic acid terminated. The magNPs were characterized using transmission electron microscopy (TEM, FEI Titan 80-300 operating at

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300 kV) and dynamic light scattering (DLS, Zetasizer-Nano, Malvern, UK).20,21 Immobilization of MMP Antibodies on magNP. The carboxy-terminated magNPs were activated to form NHS esterterminated surface by reacting with 1:1 mixture of 100 mM of N-hydroxysuccinimide (NHS, Sigma) and 400 mM of 1-(3dimethylaminopropyl)-3-ethylcarbodiimide (EDC, Fluka) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 5. The reaction was conducted in a shaker for 30 min at room temperature. The activated magNPs were then rinsed three times with MES buffer by means of a magnetic column (MiniMACSTM, Miltenyi Biotec) to capture the magNPs and remove any unreacted EDC/NHS. The activated magNPs were then modified by incubating with the desired MMPAb, MMP-1 antibody (0.2 mg/mL, R&D Systems) or MMP-9 antibody (0.2 mg/mL, R&D Systems), for 4 h at room temperature. The effect of different densities of MMP-1Ab immobilized on the magNPs was investigated. For this purpose, we prepared immunomagNPs modified with six MMP-1Ab concentrations: 0, 1.6 x 10-7, 3.1 x 10-7, 6.3 x 10-7, 1.6 x 10-6 and 3.1 x 10-6 M. After immobilization, the resulting immuno-magNPs were washed three times using phosphate buffered saline (PBS) by means of a magnetic column. Fourier transform infrared (FTIR) analysis was conducted after each surface functionalization step. FTIR spectra were obtained using a Vertex 70 Hyperion microscope (Bruker) in the reflectance mode over the range of 650-4000 cm-1, at a resolution of 22 cm-1 and averaging 64 scans. FTIR samples were prepared by placing a small drop of magNP, activated magNP or immuno-magNP solution on the surface of a p-type Si wafer. The samples were dried in a desiccator under vacuum to generate a thin magNP layer on the wafer surface before FTIR analysis. Fabrication and Functionalization of pSiRM Samples. The pSiRM samples were prepared by electrochemical etching as described in our published procedure.7 Briefly, the pSiRM samples were fabricated by anodically etching an n-type Si wafer with a resistivity of 0.008-0.02 Ω cm using a current density alternating between 50 mA cm-2 for 2288 ms and 25 mA cm-2 for 1820 ms corresponding to high porosity (HP) and low porosity (LP) layers, respectively. The active layer was etched at a current density of 50 mA cm-2 for 9152 ms resulting in a pSiRM with a configuration of (HP/LP)3(HP)4(LP/HP)3. Surface functionalization of pSiRM prior to biosensor experiments followed our previous published work.7 Shortly, the freshly etched pSiRM samples were functionalized by thermal hydrosilylation of neat undecylenic acid at 120 °C for 3 h under an argon flow. The hydrosilylated pSiRM surface was rinsed with ethanol and dried under a stream of nitrogen gas before activation to form NHS esterterminated surface. The activation was performed by reacting the hydrosilylated pSiRM samples with 5 mM NHS in water in the presence of 5 mM EDC for 20 min at room temperature. 10 mM of the fluorogenic MMP peptide substrate, DabcylGaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-Lys-NH2, was then immobilized on the pSiRM samples (Figure S-1). After overnight incubation, the pSiRM samples were rinsed with water, 2:1 water/ethanol, 1:2 water/ethanol and ethanol and then dried under a stream of nitrogen gas. Biosensor Experiments. Immuno-magNPs were used to harvest MMP in a buffer solution prepared from 50 mM Trizma base (Sigma Aldrich) pH 7.6, 150 mM sodium chloride (NaCl, Chem Supply), 5 mM calcium chloride dihydrate

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(CaCl2.2H2O, Ajax Chemical Ltd.), 1 µM zinc chloride (ZnCl2, Merck) and 0.01 % Brij L23 (Sigma Aldrich).7,10,22,23 Immuno-magNPs were incubated in buffer solution containing 1.2 x 10-12 M MMP at 37 °C for 5 min allowing the immunomagNPs to bind the MMP. Afterwards, immuno-magNPs were captured on magnetic column and washed three times using the same buffer. Immuno-magNPs were released into 100 µL Trizma-based buffer solution and incubated with the peptide-functionalized pSiRM surface for 15 min at 37 °C.7 Afterwards, the pSiRM surface was rinsed with water, 2:1 water/ethanol, 1:2 water/ethanol and ethanol to remove the magNPs and cleaved peptide fragments. The pSiRM surface was dried under a stream of nitrogen gas and placed in a fluorometer cuvette having a special holder to support the surface sample. Finally, the fluorescence intensity of EDANS was measured using a fluorometer (Perkin Elmer LS 55 Luminescence Spectrometer). The emission was measured at an excited wavelength of 340 nm, excitation and emission slit widths of 5 nm each and a scan speed of 200 nm min-1 with the angle formed by the light source and the defect layer of the pSiRM, in respect to the surface normal, set to 36° as the previously reported optimum condition.7 The effect of incubation time between magNP-MMP-1Ab and MMP-1 on sensor response was investigated. The magNP were functionalized with 1.6 x 10-7 M MMP-1Ab and then incubated in 1.2 x 10-12 M MMP-1 for 0, 2.5, 5, 10, 15, 30 or 60 min. Afterwards, the immunomagNPs were harvested and incubated on the peptidefunctionalized pSiRM surface for 15 min and the fluorescence emission of EDANS was measured. Selectivity Test in Buffer Solution. The selectivity towards MMP-1 and MMP-9 was firstly tested in buffer solution. We prepared six samples of immuno-magNPs, three of them were modified with 1.6 x 10-7 M MMP-1Ab and the other three with 1.6 x 10-7 M MMP-9Ab. Each sample of magNPs-MMP-1Ab and magNPs-MMP-9Ab was incubated in Trizma-based buffer solution7 containing 1.2 x 10-12 M of MMP-1, 1.2 x 10-12 M of MMP-9 and a mixture of 1.2 x 10-12 M MMP-1 and 1.2 x 10-12 M MMP-9. The MMP binding was done for 5 min followed by the capture of immuno-magNP on a magnetic column and their subsequent release and incubation on the peptide-functionalized pSiRM surface for 15 min prior to measuring EDANS fluorescence emission. Selectivity Test in Human Wound Fluid. Following the selectivity test in buffer solution, we also performed similar experiments in a wound fluid sample. The sample was collected from the Queen Elizabeth Hospital (Adelaide, South Australia). The study protocol, which conformed to the ethical guidelines of the 1975 Declaration of Helsinki, was approved by the Health Service Human Research Committee and Central Northern Adelaide Health Service Ethics of Human Research Committee. The presence of MMP in wound fluid was qualitatively confirmed using Western Blot analysis.23 We prepared eight samples of magNPs, four of them were modified with 1.6 x 10-7 M MMP-1Ab and the rest with 1.6 x 10-7 M MMP-9Ab. Each immuno-magNP sample (magNPs-MMP1Ab or magNPs-MMP-9Ab) was incubated in 10-fold dilution of human chronic wound fluid, 10-fold dilution of wound fluid spiked with 1.2 x 10-12 M of MMP-1, 10-fold dilution of wound fluid spiked with 1.2 x 10-12 M of MMP-9 and 10-fold dilution of wound fluid spiked with a mixture of 1.2 x 10-12 M MMP-1 and 1.2 x 10-12 M MMP-9. MMP binding to magNPs was done for 5 min prior to their magnetic capture, followed by the incubation of modified magNP on the functionalized

pSiRM surface for 15 min and measurement of EDANS fluorescence emission.

RESULTS AND DISCUSSION Our previously developed optical biosensor was based on fluorogenic peptide-functionalized pSiRM and was responsive towards MMP-1, -2, -3 and -9.7 However, the selectivity of the pSiRM biosensor towards different types of MMPs has not been achieved yet and this is important from the perspective of clinical translation since different MMPs are related to different phases of wound healing and since determining total MMP level does not provide a clear diagnostic value. Our aim here is to distinguish two different types of MMPs as proof-ofprinciple of multiparametric wound marker biosensing. Here, the immuno-magNPs were utilized as a disperse-andcollect mechanism to selectively capture MMPs from human wound fluid or other biological samples. The use of magNPs can efficiently and effectively enhance the performance of some analytical methods because magNPs have a large surface-to-volume ratio, are comparable in size to many analytes of interest, are dispersible in solution and have physical properties that can be useful to enhance signal detection.24 In order to use magNPs in this analytical method and to achieve typeselective MMP detection, MMP subtype antibodies were immobilized on the carboxy-terminated nanoparticle surface. MagNPs synthesized by effective monomer growth method25 were first functionalized by ligand exchange to introduce carboxylic acid terminal groups on their surface. This process was confirmed using FTIR spectroscopy (Figure 2).

(d)

(c)

(b) (a)

Figure 2. FTIR spectra of the magNPs before (a) and after (b) ligand exchange containing carboxy-terminal groups. Carboxyterminated magNPs after activation with EDC/NHS (c) and after immobilization of MMPAb (immuno-magNPs) (d).

Figure 2a and 2b present the IR characterization of the magNPs before and after ligand exchange, respectively. Before the ligand exchange, there were prominent peaks at 2944 and 2871 cm-1 and a weak peak at 1486 cm-1. Those peaks were assigned to the C–H stretching and C–H bending of oleic acid ligands, respectively. After the ligand exchange, a sharp peak at 1693 cm-1 and a broad band at 3400 cm-1 appeared, assigned to C=O stretching and O–H stretching, respectively, confirming successful ligand exchange. The carboxy-terminated magNPs had a particle size of 10 nm as determined by DLS and TEM (Figure 3). The small particle size enables the infiltration of the functionalized magNP through the porous layer of the pSiRM with pore

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diameters and porosities of 40–60 nm and 67% for the LP layer and 110–140 nm and 83% for the HP layer, respectively.

(a)

(b)

Figure 3. DLS (a) and TEM (b) characterization of magNPs.

The carboxylic acid groups of the magNPs can be easily activated with NHS in the presence of EDC to form succinimidyl ester that allows amide coupling to MMP antibody. ImmunomagNPs were captured on a magnetic column, instead of conventional neodymium magnet, to remove unreacted reagents and perform washing steps.24 The magnetic column is used to amplify the magnetic field, which is important to capture the well-dispersed and small particles.24 The immobilization of MMPAb on the immuno-magNP surface was confirmed by FTIR (Figure 2c and 2d). Figure 2c and 2d presents the FTIR spectra after the different surface modification steps. After activation using EDC/NHS (Figure 2c), a characteristic triplet peak corresponding to the NHS ester appeared at 1722 cm-1, 1763 cm-1 and 1817 cm-1.26,27 The bands at 1722 cm-1 and 1763 cm-1 were assigned to the C=O antisymmetric and C=O symmetric stretching vibrational mode of the succinimidyl ring, respectively, while the band at 1817 cm-1 was assigned to the C=O symmetric stretching vibrational mode and the C=O stretching vibrational mode of the succinimidyl ester.26,27 Immobilization of MMPAb on the activated magNPs via amide bond formation was confirmed by the presence of the bands at 1579 cm-1 and 1664 cm-1 which correspond to amide I and II vibrations, respectively (Figure 2d).27 For MMP detection, immuno-magNPs were incubated with MMPs for 5 min prior to their magnetic capture. The immunomagNPs-MMP were then released from the magnetic column and incubated on the functionalized pSiRM surface. The pSiRM was functionalized with a fluorogenic peptide containing EDANS as the fluorophore and Dabcyl as quencher. Active MMP bound to immuno-magNPs recognized and cleaved the fluorogenic peptide substrate, producing the immobilized substrate carrying the fluorophore while cleaving away the quencher. After incubation with immuno-magNPs-MMP, the pSiRM surface was rinsed to wash off nanoparticles and peptide fragments. The fluorescence intensity of the fluorophore attached on the pSi surface was enhanced by the photonic structure of the pSiRM. In this study, we tested two different types of MMPs, MMP-1 and MMP-9 since those two MMPs have very different roles in the wound healing process. The results presented in Figure 4 show the fluorescence emission observed on the pSiRM sensing platform incubated with either magNP-MMP-1Ab-MMP-1 (Figure 4a) or magNP-

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MMP-9Ab-MMP-9 (Figure 4b) at the same MMP concentration of 1.2 x 10-12 M. The 15 min time incubation on the functionalized pSiRM surface used in this experiment was based on the incubation time optimized in our previous published work.7 The observation of strong fluorescence emission indicates that the two MMPs bound to immuno-magNPs were still active and able to cleave the fluorogenic peptide substrate, thereby confirming that, on the one hand, the particle size of immuno-magNPs were small enough to infiltrate the porous layer of pSiRM and, on the other hand, the binding of MMP to MMPAb did not significantly impact on the enzymatic activity of MMPs. As a control experiment, the magNP-MMPAb in the absence of MMP were also incubated on the pSiRM sensing platform. No fluorescence signal was detected (dotted line in Figure 4a and 4b). This corroborates our finding that the fluorescence signal was due to the presence of MMP cleaving the MMP substrate and not due to the magNP producing a nonspecific fluorescence signal or cleaving the fluorogenic peptide in the absence of MMP. (a)

(b)

Figure 4. The full lines indicate fluorescence emission of the EDANS from fluorogenic peptide-functionalized pSiRM after incubation with magNP-MMP-1Ab-MMP-1 (a) and magNPMMP-9Ab-MMP-9 (b). The dotted lines indicate EDANS emission after incubation with immuno-magNPs in the absence of MMP.

Those experiments confirmed that functionalizing magNPs with MMPAb was an effective method to harvest MMPs from the solution without affecting the activity of MMPs. Following on from these sensing experiments, we then studied the effect of the density of MMPAb immobilized on magNPs to obtain maximum fluorescence emission during biosensing. Since the fluorescence intensities observed for MMP-1 harvested on magNP-MMP-1Ab and MMP-9 on magNP-MMP-9Ab (Figure 4) were similar, the study was only conducted for MMP-1. In order to explore the effect of the density of MMP-1Ab immobilized on magNPs, we used six different MMP-1Ab concentrations that were incubated with NHS activated magNPs: 0, 1.6 x 10-7, 3.1 x 10-7, 6.3 x 10-7, 1.6 x 10-6 and 3.1 x 106 M. Each resulting type of immuno-magNPs was incubated with 1.2 x 10-12 M of MMP-1 for 5 min before exposure to the peptide-functionalized pSiRM. The EDANS fluorescence emission emitted from the pSiRM surface after cleavage is presented in Figure 5.

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Figure 6. Fluorescence emission of EDANS from the fluorogenic peptide-functionalized pSiRM after peptide cleavage with MMP-1 bound to magNP-MMP-1Ab at different incubation times between magNP-MMP-1Ab and MMP-1; (n=3).

Figure 5. Fluorescence intensity from the fluorogenic peptidefunctionalized pSiRM after incubation with MMP-1 bound to magNPs modified with different densities of MMP-1Ab. This was achieved by varying the solution concentrations of MMP-1Ab during functionalization of NHS activated magNPs. Those solution concentrations are plotted on the x-axis; (n=3).

A conspicuous emission peak was observed for magNPs functionalized with MMP-1Ab at a solution concentration of 1.6 x 10-7 M indicating that the resulting MMP-1Ab density allowed the capture of sufficient MMP-1 to cleave the substrate and produce a detectable fluorescence emission peak. The fluorescence emission intensity scaled with increasing density of MMP-1Ab immobilized on magNPs and showed a plateau above 6.3 x 10-7 M. The density of different concentrations of MMP-1Ab attached on the magNPs was also monitored using FTIR by measuring the peak area of amide I and amide II, which represent the binding on MMPAb to magNPs (Figure S-2). The areas under the amide peaks were 0.7, 14.2, 15.6, 16.9, 17.2 and 17.5 a.u. for the MMP-1Ab concentration of 0, 1.6 x 10-7, 3.1 x 10-7, 6.3 x 10-7, 1.6 x 10-6 and 3.1 x 10-6 M, respectively, confirming the increasing density of MMPAb immobilized on magNPs. We also investigated the optimal incubation time between immuno-magNPs and MMP-1 for downstream detection in pSiRM. The concentration of MMP-1Ab used was 1.6 x 10-7 M as the lowest concentration in the previous experiment that already produced a clear fluorescence signal. Seven different incubation times with MMP-1 (at 1.2 x 10-12 M) were tested: 0, 2.5, 5, 10, 15, 30 and 60 min (Figure 6). After 2.5 min of incubation, a strong fluorescence intensity was detected from the pSiRM, indicating that MMP-1 captured by immunomagNPs was very fast. Emission intensity increased with incubation time, reaching a plateau after 5 min, suggesting magNP-MMP-1 binding saturation. This result indicates that utilization of immuno-magNPs is an effective and fast method to harvest MMPs.

After investigating the effects of density of immobilized antibody and incubation time between magNPs and MMPs, we tested the selectivity of immuno-magNPs to detect the presence of two different types of MMPs. The magNP-MMP-1Ab and magNP-MMP-9Ab were incubated for 5 min with solutions of 1.2 x 10-12 M of MMP-1, 1.2 x 10-12 M of MMP-9 or mixed MMP-1 (1.2 x 10-12 M) and MMP-9 (1.2 x 10-12 M). The immuno-magNPs with the harvested MMPs were then incubated with the fluorogenic peptide-functionalized pSiRM for 15 min and the fluorescence intensity was measured (Figure 7). The first two bars in Figure 7 present the fluorescence intensity of the EDANS from the pSiRM after peptide cleavage by MMP-1 bound to magNP-MMP-1Ab (grey-dotted bar) and magNP-MMP-9Ab (grey bar). As expected, the fluorescence intensity was significantly higher with P < 0.0001 (significance at 95% confidence) for the MMP-1 sample incubated with magNP-MMP-1Ab (17.3 ± 1.2 a.u.) compared to magNP-MMP-9Ab (1.0 ± 0.1 a.u.). The small fluorescence signal observed for the latter may be due to a small amount of MMP-1 non-specifically adsorbed to magNPs or MMP-1 trapped during the magnetic collection and washing steps.

Figure 7. Selectivity test of immuno-magNPs in buffer solution containing MMP-1, MMP-9 and a mixture of MMP-1 and MMP9; (n=3).

In the centre two bars, a similar trend is evident. The EDANS fluorescence intensity of the pSiRM upon incubation with MMP-9 bound to magNP-MMP-9Ab (grey bar) was significantly higher (25.5 ± 2.8 a.u.) than that of MMP-9 bound to magNP-MMP-1Ab (grey dotted bar) (2.3 ± 1.1 a.u.) with P < 0.0001 (significance at 95% confidence), confirming the higher affinity of magNP-MMP-9Ab for MMP-9 than for MMP-1. The two bars on the right of Figure 7 show the resulting fluorescence signals from a mixed MMP-1 and MMP-9 sample at the same concentration where MMP was captured with magNP-MMP-1Ab (grey dotted bar) and magNP-MMP-9Ab (grey bar). The average values were 14.9 ± 4.0 a.u. and 20.5 ± 1.1 a.u., respectively. These values were not significantly different from the respective values for single MMP (MMP-1: 17.3 ± 1.1 a.u. vs. 14.9 ± 4.0 a.u.; and MMP-9: 25.5 ± 2.8 a.u. vs. 20.5 ± 1.1 a.u.), with the P values of 0.475 and 0.171 (significance at 95% confidence), respectively. This result

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corroborates that in the mixture of MMPs, the magNPMMPAb were able to selectively bind the targeted MMP. The selectivity test was also conducted in human wound fluid. Sets of magNPs functionalized with MMP-1Ab or MMP9Ab were prepared as described above. Each immuno-magNP sample was incubated with a 10-fold dilution of human wound fluid or diluted wound fluid spiked with MMPs. Harvested immuno-magNPs were exposed to the pSiRM before measuring the resulting fluorescence intensity (Figure 8). EDANS fluorescence intensity emitted from the pSiRM incubated with magNP-MMP-9Ab (7.7 ± 0.8 a.u.) was higher than the intensity from the surface incubated with magNPMMP-1Ab (4.1 ± 0.2 a.u.), indicating that the wound fluid sample contained more MMP-9 than MMP-1. The mag-NPMMP-1Ab and magNP-MMP-9Ab were also incubated with wound fluid sample spiked with a concentration of 1.2 x 10-12 M of either MMP-1 or MMP-9 and a mixture of MMP-1 and MMP-9 (each component at 1.2 x 10-12 M concentration).

Figure 8. Selectivity test of magNP-MMPAb in diluted human wound fluid and human wound fluid spiked with MMP-1, MMP-9 and mixture of MMP-1 and MMP-9 (denoted as WF MMP-1, WF MMP-9 and WF mixed MMP, respectively); (n=3).

For the wound fluid sample spiked with MMP-1 (WF MMP-1), EDANS fluorescence observed on the pSiRM surface after harvesting with magNP-MMP-1Ab (grey dotted bar, 22.4 ± 2.1 a.u.) was significantly higher than the intensity from the unspiked wound fluid (4.1 ± 0.2 a.u.) with P < 0.0001 and from the surface incubated with magNP-MMP9Ab (grey bar, 6.5 ± 0.6 a.u.) with P < 0.0001. The fluorescence intensity was similar to the fluorescence signal observed after magNP-MMP-1Ab binding the MMP-1 at the same concentration in buffer solution (the first two bars in Figure 7), showing that the 10-fold diluted wound fluid does not interfere with the measurement. A similar trend was observed when magNP-MMP-9Ab were incubated with wound fluid spiked with MMP-9. The fluorescence intensity observed on the pSiRM exposed to magNP-MMP-9Ab was higher (28.6 ± 1.0 a.u.) than for magNP-MMP1Ab (3.5 ± 0.9 a.u.) with the P value of < 0.0001. The intensity was also higher than for unspiked wound fluid with P < 0.0001. If the fluorescence intensity from unspiked wound fluid sample is subtracted from the intensity for the spiked sample, then the remaining intensity signal is similar to the EDANS signal detected in buffer solution for the same concentration of MMP-9 (cf. Figure 7).

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In wound fluid spiked with mixed MMP, the fluorescence signals detected on peptide-functionalized pSiRM after incubation with magNP-MMP-1Ab and magNP-MMP-9Ab were 21.9 ± 1.6 a.u. and 28.9 ± 1.1 a.u., respectively. These intensity values did not differ significantly from those obtained upon incubation of magNP-MMP-1Ab with WF-MMP-1 (22.4 ± 2.1 a.u.), and magNP-MMP-9Ab with WF-MMP-9 (28.6 ± 1.0 a.u.) with the P values of 0.9677 and 0.9661 (significance at 95% confidence), respectively. These intensity values were also in agreement with the fluorescence intensity of mixed MMP in buffer solution after subtraction of the fluorescence intensity from the unspiked wound fluid sample. These results confirm that our biosensor can function in a complex biological sample.

CONCLUSIONS We have successfully demonstrated the proof-of-principle for type-selective biosensing of two different types of MMPs via immuno-magNPs and fluorogenic peptide-functionalized pSiRM transducers. The magNPs were modified with antibodies for specific MMP subtypes. The resulting immunomagNPs were able to bind MMP-1 and MMP-9 without affecting their enzymatic activity to cleave the fluorogenic peptide substrate. A few minutes of incubation time of immunomagNPs in a picomolar MMP solution was sufficient to produce a strong emission signal from the pSiRM, indicating that immuno-magNPs are a fast way to harvest MMP in solution. The technique allowed us to distinguish between MMP-1 and MMP-9 not only in buffer solution but also in human chronic wound fluid. Thus, our results confirm that this multiparametric sensing platform is suitable for further development as a POC diagnostic tool for chronic wound management.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental results corresponding to fluorogenic MMP peptide concentration optimization and FTIR spectra for MMP1Ab attached on the magNPs.

AUTHOR INFORMATION Corresponding Author * Nicolas H. Voelcker. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. Future Industries Institute, University of South Australia, Mawson Lakes, Adelaide, South Australia 5095, Australia. E-mail: [email protected]

Author Contributions NHV conceived the idea for this study. NHV and BPS designed the experiments. FSHK and MRD carried out the experiments. NHV, BPS and FSHK have written the manuscript. All authors have given approval to the final version of the manuscript.

Funding Sources

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Wound Management Innovation Cooperative Research Centre (WMI CRC).

ACKNOWLEDGMENT The authors thank Allison Cowin and Elizabeth Melville for providing wound fluid samples, Fran Harding for fruitful discussions about statistical calculations, Marc Cirera for the schematic illustration, the WMI CRC for providing funding for this work and top-up scholarship for FSHK and the University of South Australia for an International President’s scholarship for FSHK.

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For TOC only Type-selective detection of matrix metalloproteinases (MMPs) is achieved by using immuno-magnetic nanoparticles (immuno-magNPs) combined with a porous silicon resonant microcavity (pSiRM) biosensor. Immuno-magNPs were modified with MMP antibody specific for either MMP-1 or MMP-9 and dispersed in wound fluid to collect the corresponding MMP. The captured MMP cleaved the fluorogenic MMP substrate attached to the pSiRM resulting in strong fluorescence emission.

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