Biomaterials 35 (2014) 3729e3735

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Monitoring the VEGF level in aqueous humor of patients with ophthalmologically relevant diseases via ultrahigh sensitive paper-based ELISA Min-Yen Hsu a, b,1, Chung-Yao Yang b,1, Wen-Hsin Hsu c, Keng-Hung Lin a, Chun-Yuan Wang a, Ying-Cheng Shen a, Yu-Chen Chen a, Siu-Fung Chau d, Hin-Yeung Tsai d, Chao-Min Cheng b, e, * a

Department of Ophthalmology, Taichung General Veteran Hospital, Taichung 40705, Taiwan Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan c Institute of Nanoscience, National Chung Hsing University, Taichung 40227, Taiwan d Department of Ophthalmology, Taichung Tzu Chi Hospital, Taichung 42743, Taiwan e Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 December 2013 Accepted 10 January 2014 Available online 28 January 2014

The vascular endothelial growth factor (VEGF) level in aqueous humor has been used as an indicator to monitor specific diseases in the retinal ischemic condition. For clinical diagnosis, only about 200 mL of aqueous humor can be collected from the anterior chamber before the threat of anterior chamber collapse. It is necessary to develop an inexpensive diagnostic approach with the characteristics of highly sensitive, short operation duration, and requires small clinical sample quantities. To achieve the main objective of this study, we first prepared bevacizumab to be conjugated with HRP. We then deposited 2 mL aqueous humor from patients with different diseases onto each test zone of paper-based 96-well plates. After the colorimetric results were performed via ELISA protocol, the output signals were recorded using a commercial desktop scanner for analysis. In this study, only 2 mL from the aqueous humor of each patient was required for paper-based ELISA. The mean aqueous VEGF level was 14.4 pg/mL from thirteen patients (N ¼ 13) with senile cataract as the control. However, the mean aqueous VEGF level from other patients with proliferative diabetic retinopathy (N ¼ 14), age-related macular degeneration (N ¼ 17), and retinal vein occlusion (N ¼ 10) showed VEGF increases to 740.1 pg/mL, 383 pg/mL, and 219.4 pg/mL, respectively. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Paper-based ELISA VEGF Diagnosis Ophthalmology Diabetic disease

1. Introduction This paper describes the development of an inexpensive but robust and easy-to-handle diagnostic approach that uses a piece of filter paper to monitor the activity of three different diseasese proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), and retinal vein occlusion (RVO)eby evaluating the level of vascular endothelial growth factor (VEGF) in aqueous humor. The VEGF level in aqueous humor has been used as an indicator to monitor the activity of specific diseases in the retinal ischemic condition [1e3]. Such diseases include diabetes, which

* Corresponding author. No. 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan. Tel.: þ886 3 576 2402; fax: þ886 3 574 5454. E-mail address: [email protected] (C.-M. Cheng). 1 These authors contributed equally. 0142-9612/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2014.01.030

induces microvascular occlusion, resulting in retinal ischemia. The ischemic retina secretes VEGF, an endothelial cell mitogen and an angiogenic inducer, into the vitreous cavity. VEGF increases vascular permeability and induces the formation of new vessels arising from the plane of the retina, resulting in PDR [1]. Inhibiting VEGF secretion, when coupled with ocular drug therapy, can both decrease vascular permeability and prevent iris and retinal neovascularization in PDR, providing PDR patients with better visual outcomes via regression of iris and retinal neovascularization and reduced leakage [4,5]. In both AMD and RVO, VEGF plays a similar role in the proliferation of abnormal vessels in the retina [2,6]. Because these abnormal vessels cause severe vision loss, anti-VEGF therapies have been widely used to treat and reverse abnormal vessel developments for patients with PDR, AMD, and RVO. However, routinely monitoring VEGF concentration within the eye is hampered by insufficient sample sizes and difficult sampling techniques. There are currently two approaches for clinical VEGF

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sampling, from either vitreous or aqueous humor, to evaluate VEGF level within the eye. Both approaches are difficult to accomplish without invasive procedures, and resulting samples are small. Small sample quantities limit in-vitro diagnostics significantly, especially in regards to conventional enzyme-linked immunosorbent assay (ELISA) techniques (antigeneantibody recognition). For clinical diagnosis, only about 200 mL of aqueous humor can be collected from the anterior chamber before the threat of anterior chamber collapse. However, the clinical value of samples is especially high as VEGF is a proven biomarker of response to drug therapy [7e9]. It is necessary, therefore, to develop an inexpensive but robust and easy-to-handle diagnostic approach that is highly sensitive and specific (to specific diseases), has a short operation duration (i.e., a rapid diagnostic approach), and requires small clinical sample quantities. Such an approach would be invaluable for diagnosing specific diseases in the ophthalmology community and could possibly assist in the diagnosis of other infectious disease. Multiple bioengineering-based approaches have been developed to diagnose retinal-ischemic-based diseases such as PDR or AMD using small clinical samples including the following. First, commercial ELISA sets, which have been used to i) measure both VEGF and IL-6 (interleukin-6) levels in aqueous and vitreous samples from the same eye in order to understand whether the level of these two substances in aqueous humor is related to the severity and activity of diabetic retinopathy [7], ii) evaluate the level of VEGF, IL-6, IL-1b (interleukin-1b), and TNF-a (tumor necrosis factor-a) for potential molecular targets and pharmacokinetic analysis in aqueous samples from patients participating in drug studies or patients receiving standard care with either one or two injections of ranibizumab (lucentisÒ, Genentech, Inc., San Francisco, CA, USA) [4], and iii) study the VEGF concentration in aqueous samples before and after intravitreal injection of bevacizumab (AvastinÒ; a fully-length humanized monoclonal antibody, against all isoforms of VEGF-A) in eyes with PDR [4,5]. Second, an evidence investigator cytokine and growth factor biochip has been developed to quantitatively detect multiple analytes in aqueous humor from patients in order to determine the influence of intravitreal bevacizumab injection on the level of these cytokines and growth factors in clinically significant macular edema [10,11]. Third, Luminex xMAP suspension array technology has been used to investigate the level of 29 different growth factors and inflammatory cytokines (including VEGF and platelet-derived growth factor) in eyes with neovascular age-related macular degeneration before and during therapy with intravitreal ranibizumab injection [12]. Although these bioengineering-based approaches have allowed us to obtain promising clinically relevant information with various diseases in ophthalmology, multiple issues still need to be thoroughly addressed such as 1) the requirement of both high-cost equipment and well-trained specialists to perform either the biochip or multiplex assays, and 2) the availability of only small clinical samples obtainable only via invasive procedures. We have attempted to address the small sample volume issue with the development of P-ELISA, which only requires 2 mL of aqueous humor and has wfg/mL-level sensitivity. We also have developed a procedure to modify bevacizumab. This provides three distinct advantages: 1) it allows us to directly measure the VEGF level in aqueous humor using only a tiny amount of the modified bevacizumab as both the labeling antibody (against VEGF in aqueous humor) and the reporter (displaying the colorimetric-based output signal) in our P-ELISA; 2) it significantly increases the sensitivity of our P-ELISA, which was one of the main disadvantages of our previous study [13] and a general concern when using antibodyantigen recognition diagnostics; and, 3) it has the potential to advance research toward the co-development of drugs and

diagnostic tools to evaluate disease activity of specific diseases, i.e., the development of diagnostic approaches using modified bevacizumab to monitor the activity of three diseases (PDR, AMD, and RVO in this study). Although some investigators have proposed optical coherence tomography (OCT) imaging on an “as-needed” basis [14], P-ELISA using aqueous humor samples can provide another promising and reliable diagnostic tool. In addition, while designing the experiments in this study, we took U.S. FDA regulations into account (i.e., analytical performance and clinical validation), especially in regards to our in-vitro diagnostic approach (invitro diagnostic devices [IVD]) and performance evaluation with appropriate data analysis (potentially moving toward the codevelopment of a drug and diagnostic tool for a specific disease and approaching our ultimate goal of translational medicine). In this way, we hoped to advance an additional step from laboratorybased study to clinically relevant application, and further demonstrate strength and suitability as a transformative application in the real world that would provide impactful benefit to not just academia, but ameliorate real healthcare concerns. 2. Materials and methods 2.1. Antibodies and antigens To achieve the main objective of this study, we first prepared bevacizumab (AvastinÒ, Genentech, Inc., San Francisco, CA, USA), which is a monoclonal antibody for VEGF, to be conjugated with HRP according to the protocol of EasyLink HRP Conjugation Kit as follows: 1) dilute Avastin-antibody with HEPES (Sigma Aldrich, St. Louis, MO, USA) to the concentration of 0.8 mg/mL; 2) add EasyLink-modifier and EasyLink-HRP for 3 h at room temperature to modify the end of the Avastinantibody; 3) add EasyLink-Quencher to end the reaction. After this process, we can use this HRP-conjugated Avastin for VEGF detection using P-ELISA. Commercial human recombinant vascular endothelial growth factor (VEGF expressed in Escherichia coli, SigmaeAldrich, St. Louis, MO, USA) as a model antigen and HRPconjugated Avastin as target antibody were used to establish a calibration curve before using human aqueous humors. 2.2. Aqueous humor from patients With approval from the Taichung Veteran General Hospital Institutional Review Board, we obtained aqueous samples during cataract operation for measurement of VEGF for retinal diseases (IRB No. CF11213). VEGF has been implicated in the pathogenesis of AMD, PDR, and macular edema due to RVO. The experiments in this study were conducted from 14 aqueous samples collected from patients with PDR, 17 samples from patients with AMD, 10 samples from patients with RVO and 13 samples from patients with senile cataract as the control group. All of the 54 samplings were well tolerated by the patients with no adverse events. 2.3. Color fundus photographs and fluorescein angiography All color fundus photographs and fluorescein angiographs were performed with a confocal scanning laser ophthalmoscope, Heidelberg Retina Angiograph 2 (HRA2, Heidelberg Engineering, Germany). 2.4. P-ELISA for detection of VEGF level from patients We built a multiple-step procedure to carry out P-ELISA using paper-based 96well plates made by the wax printing method as follows [15,16]: 1) we first deposited 2 mL aqueous humor from patients with different diseases onto each test zone and allowed for 10 min of drying; 2) we aliquoted 2 mL 1% BSA blocking buffer on each test zone; 3) after 10 min of drying, we then aliquoted 5 mL HRP-conjugated Avistin with a concentration of 0.8 mg/mL to conjugate with VEGF proteins; 4) we added 2.5 mL streptavidin onto the paper-based test zones to enhance the signal at room temperature until the test zones dried; and, 5) after carrying out the washing step again with washing buffer, we placed 2 mL of a solution of the enzyme substrate (a mixture of 3,30 ,5,50 -tetramethylbenzidine and H2O2) onto our paper-based test wells to obtain colorimetric-based output signals (from colorless to blue), which were recorded using a commercial desktop scanner (EPSON; No.:GT-10000 þ) that cost w100.00 U.S. dollars. 2.5. Quantifying the intensity of test zone through a scanner The results from P-ELISA were recorded at 0, 1, 2, 4, 6, and 7 min with a handheld cellphone camera (from HTC Inc., Taiwan). The distance between cellphone and test zones, as well as exposure parameters was consistent at 0, 1, 2, 4, 6, and 7 min. The recorded color signal of each test zone after scanning was switched to grayscale at 8 bit and 600 dpi and analyzed using the commercial image-processing package software, PhotoshopÒ (Adobe, Photoshop CS5), in order to obtain the grayscale-

M.-Y. Hsu et al. / Biomaterials 35 (2014) 3729e3735 based intensity of each paper-based test zone [17]. The quantified intensity of VEGF level from each patient was repeated 20 times (n ¼ 20). If the intensity level developed in the same trend, we included this intensity in our results. If the intensity decreased or fluctuated separately with time, we excluded this intensity.

3. Results and discussion 3.1. Characteristics and calibration of paper-based VEGF test We, and others, have attempted to build low-cost in-vitro diagnostic devices (IVDs) with the following similar considerations: 1) the use of paper as the substrate (i.e., matrix-based substrate; cotton and cloth) for biochemically based reactions such as enzymatic reactions or antibody-antigen recognitions; and, 2) a commercial wax or laser printer as the manufacturing tool for the preparation of paper-based IVDs [13,15,16]. Pollock et al. first demonstrated the development and clinical testing of a paperbased, multiplexed microfluidic assay designed for rapid, semiquantitative measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a fingerstick specimen [18]. To carry out P-ELISA (as schematically described in Fig. 1a and Materials and methods), we used a wax printing method, instead of an SU-8 photolithography approach [13], to prepare paper-based 96-well plates. The calibration curve of various VEGF levels ranging from 1014 g/mL to 106 g/mL (n ¼ 8, Fig. 1b) was evaluated using the Hill equation [13]. Color intensity has an approximately linear relationship with VEGF levels without considering the blank value (Fig. 1c), and the associated color intensity of different VEGF levels is shown in Table S1. To evaluate the accuracy of our experiments, we have chosen three VEGF levels (i.e., 109 g/mL, 1010 g/ mL, and 1011 g/mL) at different dates for analyzing the coefficient

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of variation (C.V.), which is defined as the ratio between standard deviation and mean intensity. Fig. 1d displays the color intensity at different dates under three VEGF levels (109 g/mL, 1010 g/mL, and 1011 g/mL; n ¼ 8). We have defined two parameters to verify the variation, one is a within-day variation, which is the experimental result (color intensity) of one day between these different dates, and the other is a between-day variation, which is the mean value of all different dates. The results indicate that color intensity decreased with decreased VEGF levels (Fig. 1e), and the coefficient of variation at different days was within 10% (except the second day; Table S2), which indicates that accuracy would not be influenced by different testing dates via P-ELISA. Wu et al. mentioned that subtle changes can be distinguished via electronic signals, a variation that is invisible to the naked eye [19]. Therefore, we used a cellphone camera to record the colorimetric responses and analyzed their color intensities via imaging software for the following experiments. For clinical tests, we replaced VEGF antigen with sampled aqueous humor (Fig. 1a) and proceeded through the protocol. Fig. 2a represents the schematic of image recording and analysis using a cellphone camera. After aqueous humor from patients was deposited onto the test zones, we immediately captured the colorimetric results via cellphone camera after adding TMB þ H2O2 (the final step) following various reaction durations (e.g., 0, 1, 2, 4, 6, and 7 min). Following this, all colorimetric results were scanned via a high-resolution scanner, and analyzed using PhotoshopÒ to quantify color intensity (Fig. 2b). We found that the results (n ¼ 20) could be divided into three example types as follows (but not limited to): 1) the maximum color intensity was shown at the sixth minute after reaction (Fig. 2c); 2) the maximum color intensity was shown at the seventh minute after reaction (Fig. 2d); and, 3) the

Fig. 1. Schematic of P-ELISA for detection of VEGF level and calibration. (a) We first prepared a well-designed paper-based 96-well paper plate using the wax printing method. We then added 2 mL VEGF protein onto each test zone after each test zone was rinsed with 2 mL PBS. We deposited 2 mL of VEGF proteins on each test zone and then added 5 mL HRPconjugated Avistin to conjugate with VEGF proteins. After 10 min, 2.5 mL streptavidin was added to the test zone for enhancing signal readout. The color intensity was then read after adding 2 mL tetramethylbenzidine (TMB) þ hydrogen peroxide (H2O2). (b) The calibration curve of various VEGF concentrations ranging from 1014 g/mL to 106 g/mL (n ¼ 8) was evaluated using the Hill equation, I ¼ Imax ([L]n/([L]n þ [L50]n), by nonlinear regression, where Imax is the maximum color intensity (47.2  2.6), L is VEGF concentration (in U/liter), L50 is the VEGF concentration (1.4510  9.3311 U/liter) that generates a signal equal to one-half of Imax, and n is the Hill coefficient (0.25  0.02). The coefficient of determination (R2) for Hill equation is 0.9938. (c) The color intensity has an approximately linear relationship with VEGF concentration without considering blank value. (d) The color intensity at different dates under three VEGF concentrations (109 g/mL, 1010 g/mL, and 1011 g/mL; n ¼ 8). (e) The color intensity decreased as VEGF concentration decreased (between-day variation and within-day variation). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. (a) Schematic of data acquisition via a cellphone camera and data analysis via image software to perform the developing curves. (b) The colorimetric results recorded via a cellphone camera (HTC) after adding TMB þ H2O2 (the final step) at various reaction durations (e.g., 0, 1, 2, 4, 6, and 7 min), followed by scanning the colorimetric results with a scanner. After color intensity was analyzed by Adobe PhotoshopÒ, the results (n ¼ 20) could be divided into three types: 1) those for which the maximum color intensity was shown at the sixth minute after reaction in (c); 2) those for which maximum color intensity was shown at the seventh minute after reaction in (d); and, 3) those for which color intensity was irregular in (e). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

color intensity was irregular (Fig. 2e). If the intensity level developed in the same trend, we included this intensity in our results (i.e., type 1 and 2). If the intensity decreased or fluctuated separately with time, we excluded this intensity (i.e., type 3). Following this protocol, we chose the data with specific trends and analyzed samples (using scanned results) accordingly. By using serial camera photos at different durations, we could confirm that ELISA reactions evolved properly, and subtle colorimetric changes could be distinguished via electronic signals. In this study, we sought to measure the VEGF level in aqueous humor as opposed to vitreous humor. Using aqueous humor to detect cytokines via ELISA is widely used in the diagnosis of diabetic retinopathy. However, the necessity for large sample volumes (i.e., 0.1 mL per well while using plastic 96-well plates) severely hampers conventional ELISA techniques. This is a longstanding issue for many other ophthalmological disease testingeinsufficient sample volumes of aqueous humor. Although DNA and PCR amplification could be used to detect retinal ischemic conditions (i.e., Toxoplasmosis), PCR is not suitable for detecting protein-based cytokines (i.e., VEGF in this study) [20,21]. Furthermore, the sensitivity of conventional ELISA for the diagnosis of VEGF protein antigens in a buffer system was w5 pg/mL, whereas the limit of

detection of the same antigen using our P-ELISA was w33.7 fg/mL (Table 1). The sensitivity of this new bioassay was nearly 150 times higher than the conventional one for several reasons: 1) we modified the therapeutic monoclonal antibody to provide excellent Table 1 Comparisons of P-ELISA with conventional ELISA for the detection of VEGF antigens in the buffer system.

Antigen/antibody Enzyme/substrate Detection device Sensitivity

P-ELISA

Conventional ELISA

VEGF/HRP-conjugated Avistin TMB þ H2O2 Desktop scanner ($100) 33.7 fg/mL

VEGF/Avistin TMB þ H2O2 Plate reader ($20000) 5 pg/mL

Reagent/time (1) Immobilize VEGF antigen (2) BSA for blocking (3) Antibody complexing (antibody-conjugated HRP with streptavidin) (4) Signal amplification (TMB þ H2O2) Total (per test zone)

Volume (mL)

Time (min)

Volume (mL)

Time (min)

2 2 7.5

7 7 20

70 100 30

120 30 60

2

10

100

3

13.5

44

300

213

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specificity (i.e., HRP-conjugated Avistin); 2) we refined our previous ELISA protocol to eliminate the second antibody step, thus eliminating unnecessary blocking and washing steps; and, 3) we changed the enzymatic-based colorimetrical assay from alkaline phosphates (colorless to purple) to HRP (colorless to blue) of PELISA, allowing us to obtain stronger colorimetric responses. 3.2. Device performance with retinal ischemic condition clinical samples We recorded the color fundus photographs and fluorescein angiographs from patients with senile cataract, PDR, AMD, branched-RVO, and hemi-central-RVO using a confocal scanning laser ophthalmoscope (Fig. 3). The retinal fundus photographs appear normal for the senile cataract patient (Fig. 3a). However, retinal neovascularization was shown in fluorescein angiography from the patient with PDR (as indicated by arrow in Fig. 3b). From the patient with AMD, retinal neovascularization could also be seen in fluorescein angiography (as indicated by arrow in Fig. 3c). Nonperfusion area was observed in fluorescein angiography from the patient with branched-RVO (as indicated by arrow in Fig. 3d). From the patient with hemi-central-RVO, we also could observe nonperfusion area in fluorescein angiography (as indicated by arrow in Fig. 3e). After the aqueous humors from patients were aliquoted onto the test papers, we assayed for VEGF level using P-ELISA. Mean P-ELISA color intensity for VEGF from the senile cataract patient was 16.3, and the VEGF level estimated via the Hill equation was 11.8 pg/mL (Fig. 3f; n ¼ 6). The mean P-ELISA color intensity for

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VEGF from the patient with PDR was 25.3, and the VEGF level was 256.7 pg/mL (Fig. 3g; n ¼ 10). The mean P-ELISA color intensity for VEGF from the patient with AMD was 26.1, and the VEGF level was 335.8 pg/mL (Fig. 3h; n ¼ 6). The mean P-ELISA color intensity for VEGF from the patient with branched-RVO was 26.2, and the VEGF level was 348 pg/mL (Fig. 3i; n ¼ 6). The mean P-ELISA color intensity for VEGF from the patient with hemi-central-RVO was 28.9, and the VEGF level was 860 pg/mL (Fig. 3j; n ¼ 7). The mean that aqueous VEGF level was 14.4  8.5 pg/mL from thirteen patients (N ¼ 13) with senile cataract as the control (Fig. 4). However, the mean aqueous VEGF level from other patients with PDR (N ¼ 14), AMD (N ¼ 17) and RVO (N ¼ 10) increased to 740.1  267.7 pg/mL, 383  155.5 pg/mL and 219.4  92.1 pg/mL, respectively (see VEGF levels from each patient in Figs. S1eS4). The results show distinct differences (p value < 0.05) between senile cataract and other three diseases, which indicates that we can use P-ELISA to diagnose the retinal ischemic condition by evaluating the VEGF level in aqueous humor. Campochiaro et al. mentioned that it is feasible to perform serial aqueous taps for macular edema due to branched-RVO or centralRVO [8]. Multiple anterior chamber taps are feasible in the context of a clinical trial and provide aqueous samples that allow measurement of therapeutic targets or drugs. In particular, they allow pharmacokinetic and pharmacodynamic studies to be included in drug trials [22]. This will be useful for interpretation of therapeutic effects of all drugs, but may be particularly useful for monitoring drug levels after insertion of a sustained delivery device. Using aqueous sampling for VEGF detection is considered a

Fig. 3. Retinal images and developing curve of P-ELISA for VEGF levels in patients with senile cataract, PDR, AMD, and RVO. All color fundus photographs and fluorescein angiographs were performed with a confocal scanning laser ophthalmoscope. (a) Upper column shows color fundus photograph of a senile cataract patient and lower column shows normal results without any fluorescein leakage. (b) Upper column shows color fundus photograph of a PDR patient and the arrow indicates tractional retinal detachment and vitreous hemorrhage. Lower column shows neovascularization resulting in fluorescein leakage (the arrow indicated). (c) Upper column shows color fundus photograph of an AMD patient and macula area shows geographic atrophy (the arrow indicated). Lower column shows active neovascularization in upper peri-macula area (the arrow indicated). (d) Upper column shows color fundus photograph of an RVO patient and lower arcade shows area with branched retinal vein occlusion (the arrow indicated). Lower column shows the blockage of fluorescein (the arrow indicated). (e) Upper column shows color fundus photograph of a hemi-central RVO patient and upper arcade shows area with diffuse vein occlusion (the arrow indicated). Lower column shows large non-perfusion area of fluorescein (the arrow indicated). Developing curve of P-ELISA for VEGF levels in patients with (f) senile cataract (n ¼ 6), (g) PDR (n ¼ 10), (h) AMD (n ¼ 6), (i) branched-RVO (n ¼ 6), and (j) hemi-central-RVO (n ¼ 7). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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localized corneal tears due to the ultrahigh sensitivity of P-ELISA that we developed in this study. P-ELISA can provide an easy-tohandle and robust platform with great convenience and general affordability to carry out a fast VEGF assay. This fluidic-based diagnostic device, which requires only a small clinical sample, is highly sensitive, robust, easy to handle, and could provide a necessary breakthrough in efforts to monitor ophthalmic diseases using a molecularly based method. This could begin to pave the path toward similar diagnostics for different divisions of medicine. 4. Conclusions

Fig. 4. Aqueous VEGF levels in patients with senile cataract, PDR, AMD, and RVO. The VEGF values obtained for each patient were plotted on a log scale. The mean level of VEGF was significantly lower (KruskaleWallis post hoc testing) in patients with senile cataract (N ¼ 13) than in those with PDR (N ¼ 14), AMD (N ¼ 17), and RVO (N ¼ 10). Data are mean  standard deviation; *p < 0.05, indicating statistically significant differences compared with senile cataract.

valid biomarker for examining disease activity and surveillance for effect after anti-VEGF intravitreal injection. The optimal duration for dosing anti-VEGF therapy can be determined exactly through PELISA [23]. Individualized schedule for intra-vitreal injection can be applied according to the real level of VEGF through low-sample detection. Sharma reviewed various studies supporting the use of aqueous humor to validate drug signaling pathways and biomarkers in the eye [9]. To date, however, there are less standards or regulations for ocular VEGF testing [24], and P-ELISA may provide access to affordable point-of-care diagnosis of VEGF level. In addition, Balaiya et al. discovered that freezing adversely would cause aqueous samples with lower VEGF level compared with fresh samples [25]. The VEGF levels in aqueous humor may decrease due to sample degradation if doctors cannot diagnose in real-time. Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Neovascularization occurs in about 10% of patients with AMD; this complication is responsible for most (>90%) of the severe vision loss caused by this disease. VEGF-A is closely associated with the growth and permeability of neovascular vessels. Anti-VEGF monoclonal antibodies, such as bevacizumab and ranibizumab have been widely used in the last decade for treating neovascular AMD [4,26]. Overall, results across studies with large populations provide some reassurance that intravitreal injections of VEGF inhibitors has not resulted in a significant risk increase for adverse events. However, more emphasis should be placed on subgroup testing and precise evaluation with biomarkers that could evaluate adverse reactions [27,28]. Scoring systems, such as disease activity score (DAS), and clinical activity score (CAS), can provide precision to risk assessment in patients [29]. Considering signs, symptoms, and test results in patients and comparing them with past experience to arrive at a reasoned decision lies at the heart of all clinical medicine. In addition, other cytokines related to ocular disease can also use P-ELISA to monitor follow-up or prognosis of other ocular diseases [30]. We should note that tears carry a number of cytokines and growth factors secreted by the lacrimal gland as well as some cytokines that are locally produced that diffuse into the tear film from the corneal and conjunctival epithelia [31]. There is a distinct possibility that we may use P-ELISA to monitor VEGF level in

In this study, we developed an inexpensive and easy-to-handle paper-based ELISA (P-ELISA) diagnostic approach with ultrahigh sensitivity for monitoring vascular endothelial growth factor (VEGF) level in aqueous humor from 54 patients with senile cataract, proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), and retinal vein occlusion (RVO). In this study, only a minute clinical sample (2 mL/test) from the aqueous humor of each patient was required for P-ELISA. In addition, the operating duration of P-ELISA is much shorter than conventional ELISA (i.e., 44 versus 213 min). The mean aqueous VEGF level was 14.4  8.5 pg/mL from thirteen patients (N ¼ 13) with senile cataract as the control. However, the mean aqueous VEGF level from other patients with PDR (N ¼ 14), AMD (N ¼ 17), and RVO (N ¼ 10) showed VEGF increases to 740.1  267.7 pg/mL, 383  155.5 pg/mL, and 219.4  92.1 pg/mL, respectively. We have demonstrated that aqueous VEGF levels can be quantified with distinctly differing results (p value < 0.05) between senile cataract and three other diseases. Competing interests One patent filing (in U.S., Taiwan and China). Acknowledgments We would like to thank the National Science Council of Taiwan for financially supporting this research under Contract No. NSC 1012628-E-007-011-MY3 and NSC 102-2221-E-007-031 (to C.-M. Cheng). The procedure in this study has been approved by the Internal Ethical Committee of Taichung General Veteran Hospital, Taiwan (IRB No. CF11213). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.01.030. References [1] Adamis AP, Miller JW, Bernal MT, D’Amico DJ, Folkman J, Yeo TK, et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994;118:445e50. [2] Shams N, Ianchulev T. Role of vascular endothelial growth factor in ocular angiogenesis. Ophthalmol Clin North Am 2006;19:335e44. [3] Wong TY, Scott IU. Clinical practice. Retinal-vein occlusion. N Engl J Med 2010;363:2135e44. [4] Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, et al. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006;355:1419e31. [5] Sawada O, Kawamura H, Kakinoki M, Sawada T, Ohji M. Vascular endothelial growth factor in aqueous humor before and after intravitreal injection of bevacizumab in eyes with diabetic retinopathy. Arch Ophthalmol 2007;125: 1363e6. [6] de Jong PT. Age-related macular degeneration. N Engl J Med 2006;355:1474e 85. [7] Funatsu H, Yamashita H, Noma H, Mimura T, Nakamura S, Sakata K, et al. Aqueous humor levels of cytokines are related to vitreous levels and

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