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Jan 27, 2016 - ABSTRACT: We present a smartphone optosensing platform (SOP) using a digital versatile disc (DVD) diffraction grating for rapid in-fiel...
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Smartphone Optosensing Platform Using a DVD Grating to Detect Neurotoxins Li-Ju Wang, Yu-Chung Chang, Xiaoxiao Ge, Allison T. Osmanson, Dan Du, Yuehe Lin, and Lei Li* School of Mechanical and Materials Engineering, The Washington State University, Pullman, Washington 99164, United States S Supporting Information *

ABSTRACT: We present a smartphone optosensing platform (SOP) using a digital versatile disc (DVD) diffraction grating for rapid in-field detecting neurotoxins. The smartphone holder and sample holder were 3D printed for the SOP. A DVD grating is demonstrated for the first time in a low-cost miniature spectrometer on the SOP to quantify the concentrations of neurotoxins. The SOP is capable of detecting optical absorbance spectra within the entire visible spectral range from 400 to 700 nm with the spectral resolution of 0.2521 nm/pixel. We demonstrated the performance of the DVD grating compared with a commercial transmission grating on the SOP and a conventional microplate reader. Paraoxon, as the selected neurotoxin model, is assayed by two types of cholinesterase (ChE) on our SOP, respectively. Integrating a DVD grating in the SOP allows quantification of paraoxon in the range from 5 nM to 25 μM with the detection limit of 2.9 nM. In addition to the low assessed detection limit at medically relevant concentrations, the performance of SOP with DVD gratings provides new avenues for a point-of-care toxin diagnosis with time and cost savings. KEYWORDS: smartphone, spectrometer, DVD grating, neurotoxins, rapid diagnosis, point of care

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severe consequences, including abdominal cramps, neuromuscular disorders, hypotension, coma, and seizures. There are two types of ChE, acetyl cholinestearase (AChE) and butyryl cholinesterase (BChE). AChE is found mainly on red blood cell membranes, in neuromuscular junctions and in other neural synapses. BChE is produced in the liver and mostly found in blood plasma. Sufficient evidence has shown that Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is connected to AChE and BChE levels of the cholinergic system in brains.14,15 In addition, some nerve gases and pesticide poisonings affect the neuromuscular junction and cause the onset of muscle weakness and paralysis.16,17 Since ChE is highly sensitive to reaction with potent neurotoxins by combining with a residue in the active site of ChE and completely inhibiting enzymes, AChE and BChE are both reported to act as neurotoxin biosensors18,19 or as prophylactic agents against nerve gases and other poisons.20 When rapidly detecting neurotoxins early, the capability of measuring low concentration of ChE at the sub-ppb level is the primary issue. To date, optical spectroscopy has become the most promising, essential, and reliable characterization approach in almost every field of analytical, medical, life, food, and pharmaceutical science.21 However, current optical spectrometers are too bulky and heavy for integrating on miniature devices. Miniature optical spectrometers are developed for meeting on-chip needs in POC diagnosis but still have not provided clinical-level spectral resolution yet.22 A transmission

oint-of-care (POC) technology has emerged as a trend in immediate medical diagnosis especially after implementation of the Affordable Care Act (ACA).1 The key features of the ACA by 2015 specifically address in improvements in quality care and lowering costs. To achieve both, providing a low-cost, easy-to-use, and reliable platform with high sensitivity and accuracy for POC diagnosis is the crucial step. Applied to further personalized medicine, described as providing “the right patient with the right drug at the right dose at the right time” by the U.S. Food and Drug Administration (FDA),2 rapid detection, accurate quantification, and the transferability of data to doctors are in demand. Recently, mobile-health (mHealth) by smartphone technology has shown the feasibility of out-of-laboratory diagnostics and mobile data transfer.3 Some researchers have reported rapid tests to detect biomarkers,4,5 viruses,6 pathogens,7,8 antigens,9 and vitamins10 by exploiting the smartphone camera as a colorimeter and the results are deliverable online. Rapid diagnosis of POC by smartphones has been demonstrated by mainly using test trips which is low cost and simple to perform on-site, and the colorimetric results are quickly read by red− green−blue (RGB) color images.11,12 So far, however, rapid neurotoxin detection outside of the laboratory is still challenging for paper strip testing. This is because the paper strip technology does not fulfill the prerequisites of ultrahigh sensitivity, affordability, and simple readout at the clinical level. Potent neurotoxins, such as nerve gases and organophosphate pesticides, have been clinically proven to inhibit cholinesterase (ChE). ChE is a key enzyme in the cholinergic nervous system in lymphocytes and plays a crucial role in the nerve impulse transduction.13 The inhibition of ChE leads to © XXXX American Chemical Society

Received: November 3, 2015 Accepted: January 27, 2016

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ACS Sensors grating was used with a cellphone CMOS camera to perform the absorption spectrum measurement, such as an enzymelinked immunosorbent assay (ELISA).23 In addition to spectrum measurement, using photonic crystal biosensor (PCB)24 or surface plasmon resonance sensors (SPRS)25 with metal-coated nanopattern has been reported to enhance the detection sensitivity.26 Both PCB and SPRS, however, are expensive and need a time-consuming nanofabrication procedure. Since bringing the affordable single-use diagnostic devices to the market is being considered, this is a significant problem. To meet needs of both ACA and mHealth, we developed an affordable smartphone-optosensing-platform (SOP) in which a DVD diffraction grating is integrated as a miniature optical spectrometer to quantify neurotoxins. We compare the results with those obtained from a laboratory microplate reader for two irrelevant enzymes. In this study, paraoxon, as a modeled neurotoxin, is assayed by AChE and BChE, respectively. Paraoxon is one of the most potent ChE-inhibiting compounds in insecticides and can be easily absorbed through the skin. It was used as an assassination weapon by the apartheid-era South African chemical weapons program, Project Coast.27 Exposure to paraoxon leads to convulsions, vomiting, poor vision, dyspnoea, lung edema, and respiratory arrest. We present the capability of detecting paraoxon in the relevant clinical range of around 30 min (including sample preparation, measuring, and imaging capture of a set of 9 samples) using absorbance spectrum analysis. The images captured from a smartphone rear camera are analyzed by a home-built MATLAB (Mathworks, MA, USA) Graphical User Interface (GUI) program. We demonstrated the comparative accuracy and high sensitivity of an affordable miniature smartphone spectrometer. Most important, we posed a cost-saving optical design using a lowcost DVD diffraction grating and provided an in-field diagnosis platform which is as reliable as a laboratory instrument.

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Figure 1. (a) Illustration of the optical path of the SOP, (b) schematic of the assembled SOP, and (c) SEM image of a DVD grating surface.

Figure 2. Spectrum of (a) LED, (b) DI water, and (c) RhB (6 μg/mL) obtained by the SOP with a DVD grating. The light source is LED light. (d) Linear regression for RhB absorbance at 553 nm. normal direction of the surface of the grating; thus, the first-order diffraction pattern will be directly imaged in front of the smartphone camera. The smartphone is placed behind the diffraction grating. The smartphone holder is designed for tightly aligning the optical axis and diffraction grating with the rear camera of the smartphone. The diffraction grating was first fitted in a grating holder (6 mm in thickness and 20 mm in outer diameter). That grating holder was then fitted into a slot in the smartphone holder. The grating holder is rotatable in the smartphone holder. Thus, the diffraction direction can be aligned with the smartphone’s vertical direction. After alignment, the grating holder was tightened by a set screw. A cuvette holder was designed for both standard and semimicrocuvettes. The front end of the cuvette holder was designed to fit the collimate lens of the light source. The other end of the cuvette holder was designed to fit a 1-in.-diameter lens tube. The 3 mm by 9 mm window was printed inside the cuvette holder and right in front of the middle section of a cuvette. For the lens assembly, two 2-in.-long lens tubes (SM1L20, Thorlabs, NJ, USA) and one 0.3-in.-long lens tube (SM1L03, Thorlabs, NJ, USA) was used to precisely locate the lenses and pinhole. One adjustable lens tube (SM1 V05, Thorlabs, NJ, UA) was used to connect the lens tube assembly with the smartphone holder with the ability to adjust the focus. DVD Grating Diffraction Principle and Preparation. A DVD diffractive grating integrated into the SOP was demonstrated for the first time in smartphone quantitative sensing. A DVD has grooves ruled on its plastic transparent surface and diffraction is produced when light passing through the DVD grating. Light diffraction angle is described by eq 1

METHODS AND MATERIALS

Smartphone Optosensing Platform (SOP) Design. A SOP was designed based on an iPhone 5 (Apple Inc., CA, USA) smartphone with a rear camera, which equips an 8-megapixel backside-illuminated sensor (BIS) with 3264 × 2448 pixels. The SOP assembly has four parts: (1) a holder for a smartphone and the diffraction grating, (2) a diffraction grating holder, (3) a holder for positioning liquid sample, and (4) an optical tube assembly for the lenses and pinhole as seen in Figure 1b. The SOP has a tube shape, which has a total length of 240 mm. A commercial 3D printer (M200, Zortrax, Olsztyn, Poland) was utilized to print out the first three parts. A Zortrax M200 machine uses the fused deposition modeling (FDM) method which extrudes thermoplastic filaments and stacks them layer by layer. The 3D printing material was black opaque acrylonitrile-butadiene-styrene (ZABS) polymers (Zortrax, Olsztyn, Poland) for minimum light leakage. The optical path of the SOP is illustrated in Figure 1a. Two biconvex BK7 glass lenses ( f = 25.4 mm, LB1761-A, Thorlabs, NJ, USA) were used to focus the transmission light from a transparent cuvette on the diffraction grating. A 100-μm-diameter pinhole (P100s, Thorlabs, NJ, USA) was placed on the focal point of the first lens to remove unfocused scatter light. A collimated white light source (150W, A12CY, microscopenet, CA, USA) was used to illuminate the samples. In addition, other light sources such as sunlight and light-emitting diodes (LEDs) can be used. The demonstration of using a white light LED is shown in Figure 2. The noncontinuous light of a LED (Figure 2a) limits the application of measuring analytes in some wavelengths. To fully investigate the SOP, a continuous white light source was used in this work. A 3-mm-wide, 9-mm-high aperture window is designed in front of the cuvette to block stray light. The optical axis of this system is 45° tilted to the

d sin θm = mλ

(1)

where d is the grating space on DVD surface, θm is the diffraction angle of mth-order diffraction light and λ is the wavelength of light. A 16× DVD-R (4.7GB, Kodak, NY, USA) was used as the grating. The average grating period is 710 ± 19 nm, shown in Figure 1c. The DVD was first split into two pieces along the edge by using a razor blade, and the half piece with reflecting metal layer was used. The reflecting B

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ACS Sensors metal layer was then carefully removed by Scotch tape. To keep the diffraction grating as parallel as possible, a small piece of grating was cut down by scissors at the very edge section of the DVD. The coated dye on the DVD surface was removed with isopropyl alcohol (IPA). The diameter of the cut grating was about 15 mm. For comparison, a commercial transmission grating (1200 grooves/mm, GT13−12) was purchased from Thorlabs. The results will be discussed in the following section. Spectrum Calibration and Analysis. Pixel values read from the smartphone camera were converted to wavelength by using three laser pointers (405, 532, and 650 nm) and one helium−neon (HeNe) laser (632.8 nm, Melles Griot, NY, USA) to calibrate the pixel-wavelength relationship. The linear calibration line was fitted in Figure 3. The

SOP to measure the same set of RhB samples. Also, the laboratory microplate reader (Tecan Safire2, Männedorf, Switzerland) was utilized for three replicates. Assaying Procedure of Detecting Paraoxon. Procedure for Paraoxon Standard Solutions. AChE, BChE, acetylthiocholine iodide (ATCl), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and phosphate buffer saline (PBS) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). AChE, BChE, ATCl, and DTNB were prepared in pH 7.4 PBS at a concentration of 2 μg/mL, 2 μg/mL, 2 wt‰, and 0.05 wt‰, respectively. Two sets of paraoxon standard solutions were prepared in PBS. The serial concentrations of paraoxon standards were 100, 50, 20, 10, and 5 nM. Blank solutions contained 0 nM paraoxon in PBS were prepared as well. One set of paraoxon standards was assayed by AChE and the other set was assayed by BChE in the following procedure. Aliquots of 50 μL of paraoxon standards (0−100 nM) and 50 μL of ChE solution (2 μg/mL) were added separately to six microcentrifuge tubes. The tubes were vortex-mixed for 5 s and incubated for 15 min at 37 °C. During incubation, paraoxon inhibited AChE or BChE activities. Subsequently, 100 μL ATCl (2 wt‰) was added to the tube, followed by another incubation at 37 °C for 15 min. Excess AChE or BChE, which were not inhibited by paraoxon, catalyzed the hydrolysis and transform ATCl into the thiocholine. 200 μL DTNB solution (0.05 wt‰) was then added to the tube. Transformed thiocholine reacted with DTNB and generated yellow 5-thio-2nitrobenzoate anion (TNB). The yellow color was quantified by absorbance at 410 nm characterized by the SOP and the microplate reader. Procedure for Paraoxon Field Spike Solutions. To assess the suitability of the SOP, paraoxon was spiked at varying concentrations (100, 50, 20, 10, and 5 nM) into tap water collected in the laboratory at Washington State University. The field spike solutions were assayed by BChE. The field blank solution, which contained 0 nM paraoxon in tap water, was prepared to examine the interferences from tap water. Aliquots of 50 μL of paraoxon field spike solutions (0−100 nM) and 50 μL of BChE solution (2 μg/mL) were mixed and incubated at 37 °C for 15 min. After adding 100 μL ATCl (2 wt‰) and incubating for another 15 min at 37 °C, we introduced 200 μL DTNB solution (0.05 wt‰) to the tube. The yellow product was measured by the SOP and the microplate reader. Imaging Acquisition. All spectrum images were taken by the iPhone rear camera in off-exposure and autofocus modes. Five images for each sample were captured in continuous shooting mode. All images were imported to MATLAB for analysis. We built a GUI program in MATLAB for batch processing one set of images. The relationship between pixel position and wavelength were mapped first,

Figure 3. Calibration of pixel vs wavelength by three laser pointers and one HeNe laser. resolution of the SOP is 0.2521 nm/pixel. The spectrum/pixel resolution can be further improved by using a smartphone camera with a smaller pixel size and greater pixel number. However, this resolution is also limited by the camera’s lenses, the quality of the grating, and the light source. For each sample, five images were captured continuously and the average of these five images was analyzed. In spectrum analysis, a symmetry center line of the spectrum was first decided. For each wavelength point, the average of 41 pixels (20 pixels on each side) along a line, which is vertical to the symmetry center line, was used. The intensity of each wavelength point was obtained by calculating the sum of the red, green, and blue values.24 Validating the SOP by Measuring Rhodamin B. Rhodamine B (RhB, ≥95%), supplied from Sigma-Aldrich (St. Louis, MO, USA), was serially diluted in deionized (DI) water from 7.5 μg/mL to 5 ng/ mL as the samples and DI water were used as the reference. All liquid samples were filled in cuvettes (10 mm path length, Thermo Fisher Scientific, Waltham, MA, USA). For comparison, the DVD and the commercial transmission grating were separately assembled on the

Figure 4. Spectrum of (a) white light, (b) DI water, and (c) RhB (5 μg/mL) obtained by the SOP with a DVD grating. Absorbance spectra obtained by (d) the SOP with the DVD grating and (e) the microplate reader. C

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ACS Sensors as described in the Spectrum Calibration section. The five independent images of every sample were then analyzed and yielded transmittance spectrum profiles versus wavelength. These five spectra for one sample were averaged to yield a single transmittance spectrum. Transmittance (T) was converted to absorbance (A) by A = −log10 T. The batch process for every set of serially diluted samples took around 2 min.

produces perfect predictive accuracy. The R2 values are up to 0.9990 for the microplate reader and to 0.9977 for the SOP with the DVD grating. The R2 value of the SOP with the commercial grating is 0.9703 which is close to the other two methods. We can conclude that the SOP with DVD grating is able to achieve the same performance of high accuracy and precision measurements with the laboratory microplate reader. Compared with the commercial grating, the SOP with DVD grating has a higher R2 value than that of the commercial grating. It can be concluded that the DVD grating performance is comparable with the commercial grating performance. Analytical instruments produce a signal even when a blank is analyzed. This signal is referred to as the noise level. We are interested in the lowest concentration which is equivalent to the signal distinguished from background and blank noises by a particular instrument. This is the instrument detection limit (IDL) defined as the signal greater than three times the standard deviations (3σ) of the blank measures by the International Union of Pure and Applied Chemistry (IUPAC).28 In this study, the LOD was interpreted as the IDL and indicated by the dotted line in Figure 5b,c. The LOD3σ conversion from intensity unit (abs) to concentration (ng/mL) is via the sensitivity (S) of the instrument in eqs 2a and 2b

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RESULTS AND DISCUSSION Assessing the SOP by Measuring Rhodamine B. The full visible spectra from 400 to 700 nm were read out by the SOP and the microplate reader. Each sample was measured by the SOP with the DVD grating, the SOP with the commercial grating, and the microplate reader. Three spectra images of the white light source, DI water and RhB solution (5 μg/mL) obtained by the SOP with the DVD grating are presented in Figure 4a−c. It can be seen that DI water has no obvious absorbance to visible light, while RhB solution has strong absorbance at green light. The absorbance spectra of the serially diluted RhB solutions (5 ng/mL − 7.5 μg/mL) obtained by the SOP with the DVD grating are shown in Figure 4d, and the results read out by the microplate reader are shown in Figure 4e. The expanded area in low concentrations of RhB (lower that 100 ng/mL) are enlarged in Figure S1 in the Supporting Information. We examined the linearity of the absorbance at wavelength 553 nm and the concentration of RhB by three different readouts, which were the microplate reader, the SOP with the DVD grating, and the SOP with the commercial grating, shown in Figure 5a. The linear regression analysis showed that three readouts were able to perform precision measurements and the outcomes show good linearity. One way to evaluate the predictive accuracy in a linear regression model is the R-square value (R2) ranging from 0 to 1.0. R2 equal to 1.0 means the ideal linear model explains 100% of the variance and the model

S=

Δ intensity Δ concentration of analyte

LOD3σ (concentration unit) =

(2a)

3σblank S

(2b)

Determined by three replicate measurements, the statistical results of the SOP with the DVD grating and of the microplate reader were listed in Table 1. The lowest concentrations of RhB Table 1. Statistical Results of Measuring RhB by the SOP and the Microplate Reader

SOP Microplate reader

average absorbance of blank

σblank

LOD3σ (Abs)

LOD3σ (ng/mL)

R2

0.0156 0.0161

0.0002 0.0001

0.0162 0.0164

3.6 1.6

0.9977 0.9990

that can be detected, i.e., LOD3σ, on the SOP with the DVD grating and in the microplate reader were 3.6 ng/mL and 1.6 ng/mL, respectively. The reproducibility of the proposed SOP is shown in Figure S2. The standard deviation of absorbance from 9 independent measurements was 0.001 in absorbance which demonstrated that the proposed SOP is highly reproducible. By using the SOP with the DVD grating, we can achieve the IDL down to 3.6 ng/mL, which is in the detection range normally demanded by the laboratory instruments and by well-trained professionals. In the next study of assaying neurotoxins, we only demonstrated the SOP with the DVD grating and compared with the commercial microplate reader. Paraoxon Standard Solutions Assays. Paraoxon standard solutions were prepared and analyzed by the protocol described in the above section. Paraoxon inhibits ChE so that less amount of ATCl will be hydrolyzed by ChE and less DTNB will be decomposed to TNB, which generates the shallower yellow color. With the same mechanism, in the blank standard sample which contains no paraoxon, all added ChE

Figure 5. (a) Linear regression for RhB absorbance at 553 nm measured by the microplate reader, the SOP with the DVD grating and with the commercial grating. Expanded area from 5 to 100 ng/mL from (b) the SOP with the DVD grating and (c) the microplate reader. D

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Figure 6. Two sets of paraoxon standards were characterized. One set was BChE assayed and measured by the (a) SOP and (b) microplate reader. The other set was AChE assayed and measured at 410 nm by the (c) SOP and (d) microplate reader.

will completely transform ATCl to thiocholine. This maximum amount of thiocholine reacts with DTNB and yields the deepest yellow color. The blank standard sample had the strongest absorbance at wavelength 410 nm compared with other paraoxon-contained standards. In this study, we focus on the performance and sensibility of instruments. Therefore, we measured the ChE inhibition assays after 15 min incubation and did not study the kinetics of inhibition of ChE by paraoxon. The raw images were converted to absorption spectra which clearly showed the dependence on paraoxon concentrations. The results of BChE and AChE inhibition measured by the SOP and the microplate reader are shown in Figure 6. The relationships of absorption as a function of paraoxon concentration were nonlinear. The curves were fitted by a sigmoidal dose−response equation, called a four-parameter logistic (4PL) equation, governed by eq 3

f (c ) = a +

b−a 1 + (kc)s

In Figure 6, four sets of paraoxon standard concentrations were fitted to a sigmoid dose−response curve with a high goodness of fit (R2 > 99%). The dotted lines represent the signal LOD3σ. The statistic results of BChE and AChE assays of paraoxon standards measured by the SOP and the microplate reader are listed in Table 2. The curve-fitted model determined the Table 2. Statistical Results of BChE and AChE Assays of Paraoxon Standards on the SOP and the Microplate Reader

(3)

where a and b are the y-axis minimum and maximum plateau values, k is the inflection point, c represents log10 [paraoxon], and s is the Hill Slope to describe the steepness of the fitting curves. The detection limit of instrument is defined as the analyte’s signal lower than 3σ of the blank measurements (LOD3σ in intensity unit). According to IUPAC, LOD3σ in concentration unit is defined as the smallest analyte concentration required to produce a signal that is distinguishable from the signal arising from the blank solutions. We translated the IUPAC definition of LOD3σ into a mathematical form. The LOD3σ in terms of the analyte’s concentration in eq 4 was derived from eq 3 c LOD3σ = 101/ s log10(3σ /AbsBlank − 3σ − a)

average Abs of blank

σ of the blank

LOD3σ (Abs)

LOD3σ (nM)

R2

SOP Microplate reader

1.096 1.002

BChE assay 0.017 0.017

1.045 0.951

4.3 4.6

0.995 0.999

SOP Microplate reader

0.951 0.962

AChE assay 0.003 0.007

0.941 0.941

2.9 5.0

0.984 0.995

concentration LOD3σ and the measurable range between the parameters a and b. By BChE assays, the SOP is capable to detect 4.3 nM of paraoxon and is comparable to the LOD3σ of the microplate reader. On the other hand, surprisingly, the LOD3σ of the SOP to quantify the paraoxon concentration by AChE assays with 98% accuracy is 2.9 nM much lower than that of the microplate reader (5.0 nM). The main reason to achieve such a high sensitivity and the much lower concentration LOD3σ of the SOP is due to the smaller standard deviation of the SOP than that of the microplate reader. The SOP is validated with the accuracy, sensitivity, reproducibility, and the LOD. All results from measuring the standard solutions by the SOP strongly agree with these by the microplate reader. We have assessed the

(4) E

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Figure 7. Suitability study of (a) the SOP and (b) the microplate reader by detecting paraoxon concentration in field spike samples assayed by inhibiting BChE at wavelength 410 nm.

performance of the SOP in the first step and validated our SOP as the low-cost point-of-care alternative to the laboratory instruments. To our knowledge, different paraoxon biosensors lead to different detection limits and detection ranges. However, the required instruments for various biosensors are usually equipped in the laboratory. We summarized some common biosensors to detect paraoxon in Table S3 for reference purposes. Analysis of Paraoxon Field Spike Solutions. To evaluate the suitability of the SOP in real world, paraoxon was spiked into tap water as the field samples, assayed by BChE and measured by the SOP and the microplate reader. A field blank was prepared as reference. The serial dilutions were prepared from 5 to 100 nM and assayed by the described protocol. The results of absorbance at wavelength 410 nm versus paraoxon concentration in field spike samples were shown in Figure 7. The 4PL nonlinear regression model was fitted to the data and the statistic results were listed in Table 3. The accuracy of the

with those by using the laboratory microplate reader. The performance of SOP with DVD grating was also compared with a commercial transmittance grating. The white light was diffracted by both gratings and the continuous spectra were directly captured by the SOP. The linear regression of the RhB concentration with the absorbance shows that the DVD grating (R2 = 0.9977) could achieve comparable or even better performance than the commercial grating (R2 = 0.9703). With the LOD taken into account, the SOP can detect low concentrations of RhB and of paraoxon standard and spike solutions compared to the commercial microplate reader with very close to 99% accuracy. The results indicated that the performance of the SOP not only is comparable with the commercial reader, but also offers a highly sensitive and lowcost platform for point-of- care diagnosis.

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CONCLUSIONS Diffraction grating is an important optical element to obtain light spectrum. We demonstrated the first-time utilization of a DVD grating (710 nm period) as the diffraction component in a smartphone platform for quantitative assays. One DVD grating (15 mm in diameter) costs only about 2.5 cents in comparison with a $75 commercial grating (12.7 mm × 12.7 mm). The total cost of this SOP platform is about $230 and we expect the cost to be further reduced to less than $50 by using injection-molded tubes and lenses. The savings of the grating by using a DVD can greatly help us to achieve this goal. Compared to previous smartphone-based spectrum systems, this SOP system has lower cost and compatible performance for wide application in daily life. The SOPs with the DVD grating and the commercial grating were compared by measuring serially diluted RhB solutions. The analytical results exhibited good linearity. The R2 value of the DVD grating suggested that the performance of DVD grating is comparable with the commercial grating but with a much lower cost. Furthermore, we validated the performance of the SOP with the DVD grating with a laboratory microplate reader. For assessing the accuracy, standard solutions of RhB and paraoxon were analyzed. High linear regression (R2 = 0.998) and the goodness-of-fit (R2 > 0.99) were achieved in both instruments. The LOD3σ of RhB is 3.6 ng/mL for the SOP with the DVD grating and is close to that of the microplate reader (LOD3σ = 1.6 ng/mL). This SOP is able to accomplish the lowconcentration analysis the same as the microplate reader which is usually set up in the laboratory and requires welltrained professionals.

Table 3. Statistical Results of BChE Assaying Paraoxon Field Spike Solutions on the SOP and the Microplate Reader

SOP Microplate reader

average Abs of field blank

σ of the field blank

LOD3σ (Abs)

LOD3σ (nM)

R2

0.489 0.471

0.008 0.016

0.465 0.423

3.2 3.6

0.991 0.995

fitted model on the SOP is up to 99% and very close to that of the microplate reader. The LOD3σ of the SOP is 3.2 nM which is at the same detection level of the microplate reader. The field spike tests provided the strong evidence of high accuracy and low LOD3σ so that the SOP is comparatively validated. We noticed that the slightly lower values of LOD3σ in analyzing field spike solutions than that for standards. However, the values of LOD3σ of field spike solutions measured by both the SOP and the microplate reader are consistent, because BChE assays were affected by the loss rate of enzyme activity, operation errors from operators, and testing-environment conditions (e.g., temperature and air humidity). Therefore, the parallel tests of standard and field spike solutions were shown in Figure S3 to illustrate the assay performances in field spike and standard solutions at the same time. The sets of 4 parameters fitting each curve were listed in Tables S1 and S2. We aim to evaluate the suitability of the SOP and the interassay variation is negligible. Comparison of the Performance of the SOP and the Microplate Reader. The test results by using our SOP agreed F

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ACS Sensors

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The LOD of paraoxon in BChE and AChE assays indicated that the SOP has higher sensitivity than the microplate reader. While assaying field spike samples by inhibiting BChE, the suitability of SOP was comparatively validated by comparison to the microplate reader. All results strongly illustrated that the SOP is capable of measuring reliable and accurate spectra compared to the microplate reader. The performance of the SOP to achieve high reproducibility, higher sensitivity, and lower LOD than a laboratory reader provides a new potential platform which meets the clinical needs. Furthermore, to demonstrate the versatility, an LED light was used as the light source to measure the RhB solutions and the regression linearity at 553 nm achieved up to 99.5% accuracy. This affordable and customizable SOP with DVD grating is expected to fit other assaying measured by the miniature optical spectrometer in SOP. We successfully developed a costeffective compact SOP to achieve needs of both the Affordable Care Act and mHealth. A low-cost, portable, lightweight, and data-transferrable mobile sensing platform along with miniature optical spectrometer could benefit the personalized health care and point-of-care diagnostic outside laboratory with its high sensitivity for clinicians.29

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00204. Additional figures and data tables (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.:+1-509-335-4034. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Science Foundation (NSF) under Grant Number CMMI-1538439 and the Centers for Disease Control and Prevention/National Institute for Occupational Safety and Health (CDC/NIOSH) Grant No. R21OH010768 were used to support the research. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF and CDC. The authors would like to acknowledge and thank Mr. Victor Small (Washington State University) for his assistance on the 3D Printer and 3D printed parts.

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REFERENCES

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DOI: 10.1021/acssensors.5b00204 ACS Sens. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acssensors.5b00204 ACS Sens. XXXX, XXX, XXX−XXX