Collection Method of SERS Active Nanoparticles for Sensitive and

Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/ac

Collection Method of SERS Active Nanoparticles for Sensitive and Precise Measurements Javier T. Garza*,† and Gerard L. Cote†,‡ †

Department of Biomedical Engineering, Texas A&M University, College Station, Texas 77843, United States Center for Remote Health Technologies & Systems, Texas A&M Engineering Experiment Station, College Station, Texas 77843, United States

‡

S Supporting Information *

ABSTRACT: Developing surface-enhanced Raman spectroscopy (SERS) based biosensors requires not only synthesizing SERS active nanoparticles or nanoprobes that produce intense signal but also collecting them in a consistent manner to obtain sensitive and precise measurements. Nanoprobes are commonly measured in solution; however, this approach has several disadvantages that can reduce sensitivity, such as probing only a small percentage of the nanoprobes present in the sample. In this work, a novel collection device was designed, built, and tested which consistently concentrates nanoprobes in a specific area to yield highly sensitive (femtomolar) and repeatable measurements. A particular silica nanoprobe composed of aggregated silver nanoparticles with Raman reporters on them was synthesized and functionalized to measure it on the collection device. The collection device was assessed by collecting several concentrations of nanoprobes and comparing their SERS intensities to determine their limit of detection and the precision on the device. In addition, a competitive binding assay to detect cardiac Troponin I (cTnI) was used as an example to demonstrate the functionality of the nanoprobe and collection device. Nanoprobe samples (10 μL) were detected with less than 10% coefficient of variation (CV) across a range from nearly 27.4 fM to 1.7 pM using the described collection method. In the example assay, several cTnI concentrations ranging from 0 to 250 ng/mL were detected.

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metallic structures.8 Clusters of nanoparticles have been used as SERS substrates, and in order to protect and stabilize the aggregated nanoparticles functionalized with Raman reporter molecules (RRM), they have been encapsulated in silica.9,10 These silica-encapsulated aggregates can further be functionalized with biomolecules to create nanoprobes that can be used in assays.11 In SERS-based assays, functionalized nanoparticles interact with the analyte of interest and are probed to quantify the analyte concentration.12−15 One of the main challenges of SERS measurements is obtaining high repeatability at low concentrations, which is affected by measurement techniques and variations in hot spots formation.8,16 Typically, nanoprobes are measured in solution.17 However, this can be disadvantageous because (1) nanoprobes in solution can precipitate, causing them to move away from the laser focal point and consequently affect the measurement; (2) since nanoprobes are mixed in the solution, only the ones irradiated by the laser are measured, which is usually a small percentage because most Raman laser spot sizes are approximately 2 μm, and thus the probed volume is small;

urface-enhanced Raman spectroscopy (SERS) has emerged as a means by which the spectroscopic signature of molecules obtained through Raman scattering can be greatly enhanced and used for ultrasensitive detection of bioanalytes. Two primary advantages have led to the use of SERS for biosensing. First, SERS can be used to detect a very low amount of molecules, enabling the design of high sensitivity assays. Second, SERS spectral lines are narrow and thus provide the ability to multiplex and monitor several analytes using a single laser excitation wavelength.1 In order to obtain a SERS signal, a substrate that enhances the spectrum of a Raman active molecule is needed. Numerous SERS substrates have been synthesized to produce high signal enhancement and thus create more sensitive assays. Examples of substrates are silver or gold nanoparticles,2,3 nanocubes,4 nanostars,5 nanorods,6 and other structures.7 The morphology of these metallic structures and the excitation laser used can affect the degree of enhancement. Therefore, when the appropriate excitation laser wavelength is used, some substrates enhance the signal more than others. Another way to increase the SERS enhancement is by aggregating the metallic substrates to create hot spots between them. Hot spots are confined regions of intense local field enhancement produced by surface plasmon resonances that usually occur between junctions of © XXXX American Chemical Society

Received: June 15, 2017 Accepted: November 14, 2017

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DOI: 10.1021/acs.analchem.7b02318 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. Collection device. The device was built by placing a PDMS layer between two acrylic plastic slides with holes in them, making holes through the PDMS, removing the top acrylic slide, and placing 20 nm pore membranes on top of the PDMS holes. The sample was placed on top of the membrane, and vacuum was applied at the bottom of the slide.

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EXPERIMETAL SECTION Materials and Instrumentation. UVT acrylic microscope plastic slides were acquired from Ted Pella, Inc. (CA, USA). A polydimethylsiloxane (PDMS) Sylgard 184 kit was bought from Ellsworth Adhesives (WI, USA). Whatman inorganic membranes Anodisc 13 (0.02 μm pore size, 13 mm diameter) were obtained from GE Healthcare Life Sciences (PA, USA). Cardiac Troponin I (cTnI) was purchased from GenScript (NJ, USA). Cardiac Troponin I antibody [19C7] biotin was obtained from GeneTex (CA, USA). Hetero bifunctional 5 kDa PEG linker (Silane-PEG-NHS) was bought from NanoCS (NY, USA). Dynabeads MyOne Streptavidin T1 were acquired from Thermo Fisher Scientific (CA, USA). The troponin I (human cardiac specific) enzyme immunoassay test kit was obtained from GenWay (CA, USA). Hydroxylamine hydrochloride, sodium hydroxide, silver nitrate, tetraethyl orthosilicate (TEOS), (3-aminopropyl)trimethoxysilane (APTMS), ammonium hydroxide (28%), gold(III) chloride trihydrate (HAuCl4), bovine serum albumin (BSA), TRIS base, Tween 20, 5,5′dithiobis(2-nitrobenzoic acid) (DTNB), sodium chloride (NaCl), sodium phosphate, glycerol, ethanol, and isopropyl alcohol (IPA) were obtained from Sigma-Aldrich (MO, USA). The size and concentration were measured with a Nanosight Nanoparticle Tracking Analysis (NTA) system (Malvern). The zeta potential was measured on a Zetasizer Nano (Malvern). Absorbance measurements were acquired on a Tecan microplate reader. TEM images were collected on a FEI Morgagni TEM and SEM images on a JEOL JSM-7500F. A Thermo Scientific DXR Raman confocal microscope with a 532 nm laser was used to measure the SERS spectra. Samples were excited with 2 mW (unless otherwise specified), an exposure time of 1 s, and 5 exposures per reading. The estimated wavenumber resolution was approximately 5.4 cm−1 fwhm. Collection Device Fabrication. The collection device shown in Figure 1 was fabricated using acrylic plastic slides (25 mm × 75 mm), a PDMS layer, and filter membranes. First, a specified pattern of holes was drilled into two acrylic slides with a CNC machine. Then, a thin layer of PDMS (2 mm) was created by mixing 20 mL of the silicone elastomer solution with 2 mL of the curing agent, adding the mixture to a big Petri dish,

and (3) for cases in which the laser focal point can be adjusted, the lack of reference points in the solution can make it difficult to determine where to focus to perform the SERS measurement. The consistent collection of nanoprobes in a specific area for SERS measurement can address the mentioned disadvantages. In many cases, collecting nanoprobes for measurement causes the aggregation needed to produce the enhancement. In other cases, the nanoprobes are already aggregated, and the collection method only concentrates them in a specific area for measurement. Recently, efforts have focused on developing methods and techniques to allow fine control over aggregation and collection for measurement.18 Chemical methods that change the electrostatic stability of the nanoparticles have been used to induce aggregation.19 Some groups have used magnets to aggregate or collect magnetic nanoparticles in specific areas to measure their SERS signal.20 SERS substrates have also been aggregated and collected in microchannels,21 agglomerated silica particles,22 and filter membranes.23 However, the challenge of obtaining precise and sensitive measurements still exists, and improvements in the consistency of the collection methods will greatly enhance the quality of SERS measurements. Typical requirements for high performance assays usually demand detection limits at the femtomolar (fM) level or below with a precision of less than 15% CV.24,25 In this work, a unique collection device and method were developed to consistently collect nanoprobes and measure their SERS signal. A SERS active nanoprobe was also synthesized, functionalized with biomolecules to use it as an assay component, and characterized to monitor the functionalization process. The limit of detection (LoD) and precision of nanoprobes measured on the collection device were analyzed. In addition, measuring on the collection device was compared with a common measurement approach and tested with different SERS active nanoparticles. The nanoprobe and collection device were also tested in an assay to measure cardiac troponin I (cTnI) in which the nanoprobe interacted with cTnI and the unbound nanoprobe was collected to predict the analyte concentration. B


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Figure 2. Nanoprobe synthesis stages. First, Ag nanoparticles are created. Then, DTNB is added to form a SAM on the nanoparticles. Subsequently, the nanoparticles are aggregated and encapsulated in silica. After silica encapsulation, a silane-PEG-NHS linker is used to conjugate cTnI and BSA.

placed on a stirring rotator. After 30 min, an additional 25 μL of TEOS was added. The nanoparticles solution was left reacting overnight at room temperature in the rotator. Afterward, they were centrifuged and redispersed in 1 mL of ethanol. Figure 2 shows the nanoprobe synthesis approach. Protein Conjugation. The heterobifunctional linker silanePEG-NHS was used to conjugate cTnI and BSA on the nanoprobes. Cardiac troponin I was conjugated on the nanoprobes to give them functionality. To conjugate the proteins, silane-PEG-NHS (440 μL, 4 × 10−4 M in ethanol/ water 95%/5%) was added to 1 mL of the silica-encapsulated nanoparticles obtained after the differential centrifugation step described in the Supporting Information. After reacting for 1 h, the nanoprobes were washed three times using a three step process of centrifugation (2000 g, 15 min), redispersion in sodium phosphate (400 μL), and sonication. This solution was added to a mixture of BSA (23 μL, 50 mg/mL) and 10 μg of cTnI. An additional 40 μL of BSA solution was added after 15 min, and the reaction was incubated for 2 h at room temperature. TRIS buffer (550 μL, 2 × 10−2 M, pH 7.5, 0.05% Tween 20) was added afterward to quench any reactive groups left. Lastly, the nanoprobes were washed two times using centrifugation (900 g, 10 min), redispersion in 1 mL of TRIS buffer, and gentle mixing. The 1 μm magnetic beads were functionalized with cTnI antibody to use them in an assay for cTnI detection. The antibodies were conjugated by adding 20 μL of biotinylated antibody solution (2 mg/mL) to 500 μL of Dynabeads Streptavidin (2 mg/mL) and incubating them for 1 h at room temperature under gentle rotation. After this, the beads were blocked with PBS containing 0.1% BSA, washed, and resuspended in 1 mL of the PBS solution. Nanoparticle Characterization. The following measurements were collected to examine each step of the functionalization process: size, absorbance, and zeta potential. The measurements were obtained after colloid synthesis, silica

leaving it in vacuum for 50 min, and heating it at 80 °C for 45 min. A piece of the PDMS (25 mm × 75 mm) was cut and placed in the middle of the two plastic slides with their holes aligned. After that, a dispensing needle (0.009 inches inner diameter) was passed through the holes of the slides to pierce the sandwiched PDMS and create consistent holes in it. The top slide was carefully removed to avoid misaligning the PDMS layer and the bottom slide. Finally, 20 nm Anodisc filters were placed on top of the PDMS surface to cover the holes. Colloid Synthesis. Silver nanoparticles were synthesized based on the method described by Leopold and Lendl.2 Briefly, 1 mL of hydroxylamine hydrochloride (1.50 × 10−1 M) was mixed with 89 mL of sodium hydroxide (3.33 × 10−3 M) under vigorous stirring. Silver nitrate solution (10 mL, 1 × 10−2 M) was added dropwise to the stirring solution. The solution was left stirring for 15 min at room temperature. This nanoparticle solution was then used for the functionalization procedures moving forward as described below. Silica Encapsulation. The procedure to encapsulate the nanoparticles was based on the method described by Schütz.26 First, DTNB (250 μL, 1 × 10−2 M) was added to 5 mL of the synthesized silver nanoparticles to form a self-assembled monolayer (SAM) on the nanoparticles. After overnight incubation at room temperature, the nanoparticles were separated into five microtubes of equal volumes, centrifuged (2700 g, 15 min), redispersed in ethanol (900 μL) and water (65 μL), and combined in a single tube. In order to aggregate the nanoparticles and form hotspots, NaCl solution was added (175 μL, 2 × 10−2 M), incubated for 30 min, and sonicated. Subsequently, 50 μL of APTMS (0.001% in ethanol) was added to the nanoparticles and incubated for 10 min. After incubation, the nanoparticles were separated into five microtubes, centrifuged, redispersed in a mixture of IPA (1000 μL), DI water (360 μL), and ammonium hydroxide (25 μL, 28%), sonicated for 10 min, and combined in a single tube. Then, 50 μL of TEOS (1% in IPA) was added, and the sample was C


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Analytical Chemistry encapsulation, adding the PEG linker, and conjugating the biomolecules. TEM images were also acquired to inspect the nanoprobes visually. Collection Method and SERS Measurement. The SERS signal of the nanoprobe was measured on the collection device. To do this, 10 μL of the sample was placed on top of the membrane above the hole, and vacuum (approximately −0.06 MPa) was applied with a tube below the slide to suction the liquid through the membrane and hole. After the nanoprobes were collected, the collection area was focused on the Raman microscope, and an 11 by 10 point grid with a step size of 40 and 45 μm, respectively, was defined to measure the SERS spectrum at each point. The intensity peak values at 1336 cm−1, which is a peak of the Raman reporter DTNB (see Supporting Information Figure S4 and Table S1), of the measured spectra were used to plot an intensity map and averaged to obtain a value for the tested concentration. This value was also calculated using baselined spectra. For the nanoprobe measured in solution, 30 μL of the sample was added to a well. The laser was focused in the center, and one SERS measurement was obtained for each test. Assay Test for cTnI Detection. Solutions with different cTnI concentrations (0, 1, 10, 50, and 250 ng/mL) were prepared in TRIS buffer (0.05 M, 0.15 M NaCl, pH 7.6, 1% BSA, Tween 0.05%). To implement a competitive binding sequential saturation assay, 1 μL of antibody-magnetic bead solution (1 mg/mL) was added to 30 μL of the sample and incubated for 20 min. Then, 2 μL of the nanoprobe solution (3.8 × 10−11 M) was added and incubated for 10 min. Subsequently, a magnetic field was applied to separate the magnetic beads, and the supernatant was collected. Finally, 5 μL of the supernatant solution was analyzed on the collection device. To measure the samples in solution for comparison, 30 μL of each sample supernatant was probed in a well. The nanoprobes on the collection device and in solution were excited with 4 mW to obtain the assay SERS measurements.

Figure 3. Nanoprobe characterization. (A) Average diameter and (B) extinction spectra at different steps of the functionalization process.

nanoparticles in a brush conformation has been reported to be 9.8 nm,27 which agrees with the results obtained. Finally, conjugating the proteins increased the mean size of the nanoprobe by 13.7 nm to have a final size of 124.7 nm. The increase in thickness of 6.85 nm after adding the proteins is also very similar to the hydrodynamic diameter of 7.4 nm that has been reported for BSA.28 The standard deviations on each step indicated the level of polydispersity in the nanoprobe size. However, the mean size increases at each step validate the successful functionalization of the nanoprobes. The extinction spectrum was also measured at each step to determine the localized surface plasmon resonance (LSPR). The LSPR is related to the aggregation state and the interparticle gap size between nanoparticles.16 Therefore, the appearance of peaks in the LSPR spectrum can be used as an indication that the nanoparticles are in close proximity or aggregated. Figure 3B shows the different UV/vis spectra. The silver nanoparticles used had a single extinction peak at 416 nm. However, after the nanoparticles were aggregated a second peak around 575 nm was formed. The overall extinction values decreased after adding PEG and conjugating the proteins because the concentration of the nanoprobes solution decreased as particles were lost in the washing steps. Nevertheless, the two peaks were still observed after the completion of all the steps in the functionalization process indicating the stability of the silica-encapsulated aggregates. Additionally, TEM images of the nanoprobe were collected to analyze and verify the aggregation of silver nanoparticles and the silica encapsulation. In the nanoprobe synthesis process, BSA was used as a stabilizer to block spaces after conjugating the antigen and to avoid nonspecific binding. To observe the conjugated proteins on the nanoprobe surface, BSA with 3 nm gold nanoclusters was used as a marker that can be detected in the TEM images. In Figure S1 the nanoclusters surrounding the nanoprobes can be observed, verifying the attachment of BSA. The TEM images also indicate that the nanoprobes are composed of different number of aggregated Ag nanoparticles encapsulated in a silica shell with a thickness of approximately

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RESULTS AND DISCUSSION Collection Device. The device was fabricated to consistently collect and aggregate functionalized nanoprobes and measure their SERS signal. The collection slide has holes and an aluminum oxide membrane with 20 nm pores. Since the membrane adheres to PDMS and PDMS to the acrylic slide without any adhesive, a PDMS layer was placed between the slide and the membrane to seal the membrane around the holes and avoid any liquid to be suctioned at any other point but the holes. The collection slide was built by drilling 457.2 μm holes in an acrylic slide, punching holes of similar size in the PDMS layer, and stacking the membrane on top of the PDMS layer. A CNC machine was used to drill the holes in the slide in a repeatable manner. The main priority was to consistently create sealed holes with the same diameter. Nanoprobe Characterization. The size of the nanoprobe was monitored at different steps of the functionalization process with a Nanoparticle Tracking Analysis (NTA) system to verify the conjugation. The results are shown in Figure 3A. As can be observed, the silver nanoparticles had a mean diameter of 56.8 nm. These nanoparticles were aggregated and encapsulated in silica. The mean size of these silica nanoprobes was 91.5 nm. A 5 kDa PEG linker was attached to the silica to link the proteins to the nanoprobe, which increased the nanoprobe overall mean size by 19.5 nm, or rather, the shell thickness by 9.75 nm. The length of a 5 kDa PEG chain on D


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Figure 4. Assay to detect cTnI. In the assay, magnetic beads coated with cTnI antibodies are mixed and incubated with the sample to be tested. A known amount of nanoprobe is added, and after incubation, a magnetic field is applied to separate the beads from the free nanoprobes in the supernatant. Finally, the nanoprobes in the supernatant are collected on the collection device to measure their SERS signal.

20 nm. The nanoparticles were purposely aggregated to create hot spots between them and make a nanoprobe that can produce a high SERS signal. A glycerol-based differential centrifugation method (described in the Supporting Information) was used to separate the silica-encapsulated nanoparticles with similar weight and reduce the level of polydispersity. Additional nanoprobe characterization tests included measuring the zeta potential (Figure S2) and using an ELISA kit to determine the amount of active cTnI attached to the nanoprobe (Figure S3). Using the Nanoprobe and Collection Device in cTnI Assay. A sequential saturation competitive binding assay to detect cTnI was implemented with the nanoprobe functionalized with cTnI and magnetic beads functionalized with cTnI antibodies. The SERS measurements were performed on the collection device. The assay procedure can be observed in Figure 4. First, the sample to be measured is added to a solution with magnetic beads, where the cTnI of the sample binds to the antibodies on the beads. After 20 min of incubation, a known amount of nanoprobe is introduced, which binds to the available antibody binding sites that are not occupied by the sample cTnI. The components are incubated for an additional 10 min. Then, a magnetic field is applied to separate the magnetic beads and the free nanoprobes in the supernatant. A volume of this supernatant is then applied to the collection device to concentrate the nanoprobes on a spot and measure their signal. Each nanoprobe functionalized with DTNB has a specific SERS signal when it is probed. Therefore, when the nanoprobes are collected on the collection device the measured SERS intensity is proportional to the amount of nanoprobes present, which is proportional to the amount of analyte in the sample. Since a known constant amount of nanoprobes and magnetic beads is used in each assay test, the concentration of an unknown sample can be determined if the measured signal is compared to a calibration curve. Testing the Nanoprobes in the Collection Device. Solutions with known concentrations of nanoprobe were tested in the collection device to determine their SERS signal. Figure 5 shows the SEM images of the collection device with nanoprobes collected on the membrane. As can be observed, the nanoprobes are collected only on the areas in contact with the holes. When a low concentration of nanoprobes is passed through the membrane, they are spread throughout the area

Figure 5. SEM images of nanoprobes aggregated on the collection device. Low concentration: (A), (B) zoom. Medium concentration: (C), (D) zoom. High concentration: (E), (F) zoom.

and the pores can be observed in the SEM image (Figure 5A, 5B). However, when a higher concentration of nanoprobes is analyzed, they cover all the area and form a thicker layer as they are stacked on top of each other (Figure 5E, 5F). The main function of the collection device is to gather together the nanoprobes in a small area and have the amount of nanoprobes as the only changing variable in a SERS measurement. An effort was made to make the holes consistently and keep the aggregation area as similar as possible for each measurement. After the nanoprobes were gathered with the collection device, the area was focused in the Raman microscope, and the SERS spectrum was measured at defined points covering the collection area. SERS maps were plotted by using the intensity at 1336 cm−1 of all the spectra acquired. Figure 6 shows the E


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Figure 6. SERS intensity maps of different concentrations of nanoprobe collected on the collection device. To plot a map, the SERS spectrum was measured at different points covering the collection spot (11 × 10 points, 40 and 45 μm step size, respectively). Then, the peak value at 1336 cm−1 of each spectrum was plotted for the corresponding grid point.

maps of the SERS intensity at 1336 cm−1 as different amounts of nanoprobe were analyzed on the collection device. As can be observed, the signal is mainly detected on the holes of the device. The signal increases with increasing concentration and reaches a maximum at 7.03 pM. At the blank measurement no signal is observed. The intensities at the specific wavenumber of interest of all the spectra measured in the grid to plot each map were averaged to obtain a single value for each collection spot. These values were then plotted with their corresponding concentration of nanoprobe used when 10 μL was tested. Three different measurements with the same amount of nanoprobe were tested to calculate the standard deviation. To compare the measurement on the collection device with a common measurement method, 30 μL of the same nanoprobe concentration was measured in a well using the same instrument settings. Figure 7A shows the concentration curves of the nanoprobe determined on the collection device and in solution. As can be noted, using the collection method allows to detect lower concentrations than measuring in solution (more than an order of magnitude lower concentration). This occurs because the nanoprobes of the sample are concentrated in a specific area, and thus more of them are probed. Utilizing the collection device has additional advantages, such as facilitating the laser focus on the collection area and employing less sample volume. It is expected that measuring SERS active nanoparticles that are not already aggregated, unlike the silica nanoprobes used, would result in less sensitive measurements if measured in solution. As can be observed in the logarithmic plots, portions of the curves can be used to calculate a linear function to predict the concentration. In the collection device curve, at higher concentrations the SERS signal flattens out and on average appears to decrease slightly but is effectively flat given the increase in standard deviation. This standard deviation increase is likely due to the membrane saturating. Therefore, the results obtained suggest that the collection device can be used to measure a specific range of concentrations, from the LoD to the saturation point. Concentrations above the saturation point could be measured in solution. Comparison of Nanoprobes with Ag-DTNB Nanoparticles. The SERS signal concentration curve of the nanoprobe on the collection device was also compared with

Figure 7. Concentration curves. (A) Comparison of signals obtained when the same concentrations of nanoprobe were measured on the collection device and in solution. For measurements on the collection device, each point is the mean of the SERS intensity values at 1336 cm−1 probed at different points on the collection area when 10 μL of the corresponding nanoprobe concentration was analyzed. (B) Comparison of signals obtained when the same concentrations of nanoprobe and Ag nanoparticles functionalized with DNTB were measured on the collection device.

the SERS signal concentration curve of Ag nanoparticles functionalized only with DTNB (Ag-DTNB). The same concentrations were used to test them in the collection device. As can be noted in Figure 7B, the minimum concentration of nanoprobe that can be detected is about an order of magnitude lower than the concentration of Ag-DTNB nanoparticles. The LoD of the nanoprobe and Ag-DTNB nanoparticles measured on the collection device was calculated to be 16.2 and 517 fM, respectively, using the raw spectra (see Supporting Information F


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1.76 pM or higher were less than 10% when baselined data was analyzed (see Figure 8A and Supporting Information Table S7). The CVs of the Ag-DTNB nanoparticles were more variable, with most CV values of less than 10% obtained for concentrations higher than 0.43 pM (see Figure 8B and Supporting Information Table S4 and Table S5). This indicates that measuring the nanoprobe with the collection device is the best of the three options considered to measure low concentrations with high repeatability. Assay Results. The SERS values obtained for the concentrations of cTnI tested in the assay are presented in Figure 9. As can be observed, SERS intensities increase with

Table S8). All the collected spectra were also baseline subtracted using an algorithm based on a method described by Ning29 before averaging the intensities at 1336 cm−1 to obtain a value for each concentration. The LoD calculated with the baselined data improved to 12.9 fM for the nanoprobe and 190 fM for the Ag-DTNB nanoparticles. The lower LoD of the nanoprobe can be attributed to the consistent enhancement produced even after the nanoprobes are spread on the collection area at low concentrations. In each nanoprobe there are about two or more silver nanoparticles encapsulated in silica that interact with each other to form hotspots and enhance the signal of the DTNB molecule. In comparison, all the Ag-DTNB nanoparticles are aggregated on the membrane to form less controlled hotspots as they are not encapsulated. When the concentration is low, these nanoparticles spread on the collection area, and less or no hotspots occur. Coefficient of Variation Comparisons. The coefficient of variation (CV) was calculated for the triplicate measurements of nanoprobe and Ag-DTNB nanoparticles gathered with the collection device to determine how precise this method can be. In addition, the CVs of the nanoprobe in solution measurements were calculated for comparison. As can be observed in Figure 8, for nanoprobe concentrations between 27.4 fM and

Figure 9. Assay response to different concentrations of cTnI tested. Triplicate samples were measured on the collection device and in solution for comparison.

increasing cTnI concentrations for the samples measured on the collection device. In contrast, low intensities with no significant differences between the cTnI concentrations tested are observed for the samples measured in solution. This occurs because of the low amount of nanoprobe present at these concentrations, which could not be detected in solution without concentrating the nanoprobes on the collection device. For measurements in solution all the samples had a CV higher than 30%. Alternatively, on the collection device, the samples at 10 ng/mL or higher had a CV of less than 15%. This suggests that assay optimization would be necessary to detect lower concentrations with better precision if required. It is important to note that the assay was not optimized to obtain the response that would cause the greatest signal difference between the minimum and maximum concentrations analyzed but as an example assay for demonstrating the ability of the collection device.

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Figure 8. Coefficient of variations (CVs) of three replicate measurements at different concentrations of (A) nanoprobe measured on the collection device and in solution and (B) nanoprobe and AgDTNB measured on the collection device. Baselined spectra were used to calculate the values.

CONCLUSIONS A method to fabricate a collection device to consistently gather nanoprobes and measure their SERS signal was demonstrated and tested. In addition, a nanoprobe was synthesized, characterized at different stages of its functionalization process, and used to test the collection method. Different concentrations of the nanoprobe were measured on the device, and its LoD was determined to be 12.9 fM for the settings used. The repeatability of triplicate measurements was also analyzed. CVs were found to be less than 10% for the majority of the nanoprobe concentrations tested. The nanoprobe was also

1.76 pM measured on the collection device, their CVs were less than 10% when baselined data was analyzed (see also Supporting Information Table S3). At higher and lower concentrations, their CVs were greater than 10%, and this can be attributed to the previously mentioned saturation for the higher concentrations and approaching the LoD for the lower concentrations. For measurements in solution, only the CVs at G


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measured in solution to compare it with measurements obtained with the collection method. Furthermore, similar concentrations of Ag nanoparticles functionalized with RRM were tested and analyzed on the collection device. Based on the results obtained, it was determined that measuring the nanoprobe with the described technique resulted in lower LoDs and higher precision at lower concentrations. Finally, an assay for cTnI detection was developed to show an example of the utility of the nanoprobe and collection device. This example demonstrated how using the collection technique allowed the measurement of low nanoprobe concentrations in a precise manner, which is important for biosensor design.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02318. Additional experimental and characterization information; troponin I on nanoprobe calculations; nanoprobe SERS spectrum; experiments tabular data; LoD calculations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Javier T. Garza: 0000-0002-2559-8315 Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the support of the SLOAN Foundation Fellowship, the Texas A&M University Graduate Diversity Fellowship, and private industry support.

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REFERENCES

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