Article pubs.acs.org/ac
Facile Detection of Troponin I Using Dendritic Platinum Nanoparticles and Capillary Tube Indicators Sanghee Lee, Donghoon Kwon, Changyong Yim, and Sangmin Jeon* Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 790-784, Republic of Korea ABSTRACT: A facile method was developed for the detection of Troponin I (TnI) using dendritic platinum nanoparticles and capillary tube indicators. Dendritic platinum nanoparticles were functionalized with TnI antibodies, which were used to capture TnI in human serum. The captured TnI was conjugated to the inner surface of a glass vial, to which a hydrogen peroxide (H2O2) solution was added. After the glass vial was sealed with a screw cap containing a silicon septum, a capillary tube containing a drop of ink was inserted through the septum. The catalytic dissociation of H2O2 to water and oxygen increased the pressure inside the glass vial and raised the ink level in the capillary tube. The ink level increased with the platinum nanoparticle concentration, which is proportional to the TnI concentration. The sensitivity of this assay for TnI in human serum after a 5 min dissociation reaction, detected with the naked eye, was 0.1 ng/mL, which was better than the sensitivity of the conventional colorimetric method using the TMB oxidation reaction under the same experimental conditions. A control experiment using alpha-fetoprotein, interleukin-5, and C-reactive protein revealed that the developed method was highly selective for the detection of TnI. require bulky instrumentation. In contrast, the lateral flow immunoassay (LFA),10,11 which does not require any read-out instrumentation, has been used as a point-of-care platform; however, it has the critical disadvantage of low sensitivity (∼1 ng/mL). The sensitivity of the LFA could be improved by labeling with fluorescent or luminescent dyes, but this would require bulky and expensive detection systems.12,13 We demonstrate here a facile and sensitive method for measuring TnI concentrations using an alcohol thermometerlike glass vial and dendritic platinum nanoparticles. In this method, TnI in human serum was captured by antibodyfunctionalized dendritic platinum nanoparticles, and then conjugated to the inner surface of a glass vial functionalized with TnI antibodies. After addition of a hydrogen peroxide (H2O2) solution, the vial was sealed with a capillary tube containing a drop of ink. The pressure increase inside the glass vial caused by the catalytic dissociation of H2O2 to H2O and O2 increased the ink level in the capillary tube. This increase, detected by the naked eye, was used to determine the TnI concentration. The detection limit of the assay was found to be 0.1 ng/mL of TnI in human serum after a 5 min dissociation reaction.
L
iquid-in-glass thermometers, such as alcohol and mercury thermometers, are frequently encountered in our daily lives at home, in hospitals, and in school laboratories. In these devices, the volume of alcohol changes with temperature, and the resulting alcohol level is easily converted to temperature using an inscribed scale in the capillary tube. Although state-ofthe-art temperature measurement devices such as remote infrared detectors are available at reasonable prices, alcohol thermometers are still widely used owing to their many advantages, such as low cost, no need for external power, high reliability, and ease of use. The elegance of an alcohol thermometer lies mainly in its unique structure, consisting of a relatively large glass bulb containing red-colored alcohol and a narrow capillary tube connected to the glass bulb. This structure amplifies the small change in the alcohol volume inside a glass bulb, translating it to a large change in the alcohol level inside the capillary tube. This simple method for signal amplification may be utilized to measure biomarkers such as Troponin. Troponin is a complex of three subtype proteins: troponin I (TnI), troponin C, and troponin T. Troponin is considered a specific marker for myocardial damage because its concentration in blood increases upon damage to cardiac muscles. A number of immunoassays have been developed for measuring Troponin concentrations using microresonators,1,2 surface plasmon resonance (SPR),3−5 surface acoustic wave (SAW) sensors,6 and electrochemical sensors.7−9 However, these methods are not practical for most applications because they © 2015 American Chemical Society
Received: March 9, 2015 Accepted: April 20, 2015 Published: April 20, 2015 5004
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
Article
Analytical Chemistry
Figure 1. Schematic illustration of the detection of TnI using glass vials and capillary tubes with an ink indicator.
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Detection of TnI Using Decomposition of H2O2. Figure 1 shows a schematic illustration of the experimental procedure for the detection of TnI using an antibody-functionalized glass vial and a capillary tube containing a drop of red ink (safranin in an aqueous solution). First, 20 μL of a 2 mg/mL solution of antibody-functionalized platinum nanoparticles was added to 400 μL of TnI-spiked human serum. After incubation for 10 min at room temperature with gentle shaking, the sample containing TnI−Pt complexes was added to a gastight glass vial whose inner surfaces were functionalized with TnI antibodies. After incubation for 10 min at room temperature to allow TnI− Pt complexes to bind onto the glass surfaces, the glass vial was washed several times with 0.1% Tween 20 and PB. A 34.5% H2O2 solution was added to the sample, and the vial was sealed with a twist cap containing a silicon septum. A capillary tube containing a drop of ink was then inserted through the septum. The catalytic dissociation of H2O2 to H2O and O2 increased the pressure inside the glass vial, causing the ink level in the capillary tube to rise. The resulting rise was monitored with the naked eye or using a digital camera. To compare the sensitivity of the developed assay with the conventional colorimetric method based on the TMB oxidation reaction, we added TMB to the solution to induce a color change.
EXPERIMENTAL SECTION Materials. Chloroplatinic acid hexahydrate, 1 M sodium hydroxide solution, ascorbic acid, 3-aminopropyltriethoxysilane (APTES), glutaraldehyde, TCEP (tris(2-carboxyethyl)phosphine), Tween 20, 3,3′,5,5′-tetramethylbenzidine, and bovine serum albumin (BSA) were purchased from SigmaAldrich (St. Louis, MO, USA) and used as received without further treatment. TnI antigen and monoclonal antibodies against TnI (4T21 560 and 4T21 19C7) were purchased from Fitzgerald Industries (Acton, MA, USA) and Abcam HyTest Ltd. (Turku, Finland), respectively. Deionized water (18.3 MΩ· cm) was obtained using a reverse osmosis water system (Human Science, Korea) and used for making phosphate buffer (PB). Hydrochloric acid and H2O2 were purchased from Samchun (Pyeongtaek, Korea), and ethanol and methanol were purchased from Merck (Darmstadt, Germany). Glass vials (2 mL) and plastic screw caps with silicon septa were purchased from Agilent Technologies (Santa Clara, CA, USA). Preparation of the Capture-Antibody-Immobilized Glass Vial. A glass vial was cleaned by soaking in a mixture of hydrochloric acid and methanol for 1 h and then rinsed several times with methanol and water.14 After drying under a nitrogen flow, the glass vial was treated with 1% APTES in ethanol for 12 h to produce amine groups on the inner surface of the vial. A 1 mL, 4 μL/mL TnI antibody (4T21 560) solution was added to the glass vial, and the glass vial surface was functionalized with the antibodies using glutaraldehyde as a cross-linker. The glass vial surface was then blocked with a mixture of 1% BSA and 1% Tween 20 at 4 °C for 6 h to prevent nonspecific binding. Preparation of Detection-Antibody-Functionalized Platinum Nanoparticles. Dendritic platinum nanoparticles were synthesized by mixing 20 mL of 1.45 mM H2PtCl6, 250 μL of 1 M ascorbic acid, and 150 μL of 1 M sodium hydroxide.15 After the solution was heated at 60 °C for 10 min, the solution color changed from pale yellow to brown. The synthesized platinum nanoparticles were purified several times by centrifugation at 14000 rpm for 5 min. A half-fragment detection antibody (4T21 19C7)16 for easy immobilization of the antibody onto platinum nanoparticles with the desirable orientation was obtained by adding 10 μL of 5 mM TCEP in PB to 400 μL of PB containing 10 μg of detection antibody and incubating for 1 h at room temperature. After immobilization of the half-fragment antibody on platinum nanoparticles via Pt−S bonds, the functionalized nanoparticles were rinsed with PB solution several times, then 0.1% Tween 20 and sucrose were added to the solution to prevent nonspecific binding.
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RESULTS AND DISCUSSION Figure 2a,b show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images, respectively, of dendritic platinum nanoparticles. The average size of the dendritic nanoparticles was ∼40 nm, each of which consisted of tens of ∼5 nm platinum nanoparticles. The inset of Figure 2b shows the high-resolution TEM image of the dendritic platinum nanoparticles, indicating that the platinum consists of crystalline nanoparticles. Addition of the platinum nanoparticles to a H2O2 solution induces the dissociation of H2O2 molecules into H2O and O2. Natural enzymes such as catalase may be used for the dissociation of H2O2 molecules, but their activities are degraded over time; thus, they are not suitable for long-term storage.17,18 Because the catalytic activity of platinum nanoparticles is substantially affected by nanoparticle surface area, dendritic platinum nanoparticles were used in this experiment instead of single, large platinum nanoparticles.19−23 The X-ray diffraction (XRD) pattern for the platinum nanoparticles on a silicon wafer (Figure 2c) confirms that the dendritic platinum consists of crystalline nanoparticles. Figure 2d compares the changes in ink level during the catalytic dissociation of H2O2 caused by the dendritic platinum nanoparticles with that caused by conventional platinum nanoparticles synthesized by different methods.24,25 The results 5005
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
Article
Analytical Chemistry
Figure 2. (a) SEM and (b) TEM images of dendritic platinum nanoparticles. The average size of the platinum nanoparticles was ∼40 nm. The high-resolution TEM image in the inset of panel b indicates that the dendritic platinum consists of crystalline nanoparticles. (c) XRD pattern of synthesized dendritic platinum nanoparticles. (d) Time-dependent changes in ink level due to the dissociation of H2O2 by dendritic platinum nanoparticles (red circle) and conventional platinum nanoparticles (black square).
Figure 3. (a) Optical images of the ink level at various concentrations of TnI at 0, 1, 2, 3, 4, and 5 min after addition of H2O2 to the glass vial. The spacing between two adjacent lines is 5 mm. (b) Time-dependent changes in the ink level and the concentration of the produced O2 at various TnI concentrations: ■, 0 ng/mL; •, 0.1 ng/mL; ▲, 0.5 ng/ mL; ▼, 1 ng/mL; ◀, 10 ng/mL; ▶, 100 ng/mL. The right y-axis shows the molar concentration of O2 produced by the dissociation of H2O2.
showed that dendritic platinum nanoparticles induced far larger increases in the ink level than conventional platinum nanoparticles, despite the fact that the number, concentration, and size of the particles were nearly identical. Figure 3a shows optical images of the time-dependent changes in ink level as a function of TnI concentration. The spacing between two adjacent lines is 5 mm, and each snapshot was obtained at 0, 1, 2, 3, 4, and 5 min after the addition of H2O2 to the glass vial containing TnI−Pt complexes. Negligible changes were observed for 0 ng/mL TnI, indicating that platinum nanoparticles did not bind nonspecifically to the glass vial surface. The level of ink increased with increases in TnI concentration and reaction time, and the change in ink level could be clearly detected with the naked eye. The change in ink level for the 100 ng/mL TnI solution after a 5 min reaction was 10.6 mm, which was 8 times larger than that for a 0.1 ng/mL TnI solution. Figure 3b shows the time-dependent changes in ink level and the molar concentration of O2 produced as a function of TnI concentration. The change in level can be converted to the volume change by simply multiplying the inner diameter of the capillary tube (1.8 mm) by the change in level. Assuming that the volume change is induced solely by the O2 produced by catalytic dissociation of H2O2, changes in the ink level can be converted into the molar concentrations of the produced O2 (Figure 3b, right y-axis). The level of ink (i.e., the O2 concentration) increased almost linearly with reaction time, indicating that the catalytic production of O2 by platinum nanoparticles is a zero-order reaction, because the rates are independent of substrate concentration (i.e., H2O2 concentration). Zero-order reactions are typically observed for enzyme-catalyzed reactions in which the concentration of enzyme (i.e., platinum nanoparticle) controls the reaction rate. In zero-order kinetics, the reaction rate is dependent on the enzyme concentration, and the derivative of the product
concentration as a function of time (i.e., the slopes of the linear lines fitting the data) corresponds to the rate constant. Figure 4 shows changes in the ink level as a function of TnI concentration in serum after the catalytic dissociation of H2O2
Figure 4. Changes in ink level as a function of TnI concentration after the catalytic dissociation of H2O2 for 5 min.
for 5 min. The ink level increased linearly with the log of TnI concentration. Based on the three-sigma method, the detection limit of the assay with the naked eye was found to be 0.1 ng/ mL of TnI in human serum after a 5 min catalytic dissociation reaction, a limit suitable for the diagnosis of cardiac problems. Because the ink level increases with reaction time, the detection limit could be improved further by increasing the reaction time. A series of control experiments was conducted to examine the selectivity of the assay. After TnI antibody-functionalized 5006
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
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Analytical Chemistry
Figure 5. (a) Changes in ink level measured for human serum spiked with various proteins at 100 ng/mL. (b) Optical images of cuvettes containing different concentrations of TnI after a 5 min TMB oxidation reaction. (c) Changes in absorbance of the solutions as a function of TnI concentration.
Notes
dendritic platinum nanoparticles were dispersed in human serum spiked with 100 ng/mL α-fetoprotein (AFP), interlukin5 (IL-5) or C-reactive protein (CRP), they were allowed to bind onto the glass vial surface functionalized with anti-TnI antibodies. Figure 5a shows that changes in the ink level for AFP, IL-5, and CRP after the catalytic dissociation of H2O2 for 5 min were far smaller than that for 100 ng/mL TnI, indicating that the detection of TnI in human serum was highly selective. In addition, the sensitivity of the developed assay was compared with that of the conventional TMB oxidation reaction by platinum nanoparticles. Figure 5b shows optical images of cuvettes containing different concentrations of TnI after a 5 min TMB oxidation reaction. The color of the solution became blue as the concentration of TnI increased owing to the catalytic oxidation of TMB to tetramethylbenzidine diimine by platinum nanoparticles in the presence of H2O2. The resulting production of tetramethylbenzidine diimine causes the solution to turn blue. The detection limit of the assay with the naked eye was 10 ng/mL of TnI in human serum, a limit 2 orders of magnitude lower than that of our method under the same experimental conditions. The concentration of TnI was also determined quantitatively using a UV−vis spectrometer by measuring absorbance at 370 nm. Figure 5c shows the changes in absorbance as a function of the concentration of TnI. The detection limit with the spectrometer was found to be 1 ng/mL, still lower than that of our method with the naked eye. The higher sensitivity of our assay is attributable to the production of bubbles instead of a chemical product. Because the gas volume is 1000 times larger than the liquid volume with the same number of molecules, the volume change due to the production of hydrogen is much greater than the color change due to the production of tetramethylbenzidine diimine.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by a National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2014R1A2A2A01007027).
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CONCLUSION In summary, we have developed a facile method for the detection of TnI in human serum using antibody-functionalized dendritic platinum nanoparticles and a capillary tube containing a drop of ink. The pressure increase caused by the catalytic dissociation of H2O2 by platinum nanoparticles induces an increase in ink level, which is easily identified with the naked eye. The detection limit of the assay was 0.1 ng/mL TnI in human serum after a 5 min catalytic reaction. Because the ink level increases further with reaction time, the limit of detection could be improved by increasing the reaction time. This simple and cost-effective method may be especially beneficial for people in underdeveloped countries.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*S. Jeon. E-mail: [email protected]. 5007
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
Article
Analytical Chemistry (20) Lacroix, L.-M.; Gatel, C.; Arenal, R.; Garcia, C.; Lachaize, S.; Blon, T.; Warot-Fonrose, B.; Snoeck, E.; Chaudret, B.; Viau, G. Angew. Chem. 2012, 124 (19), 4768−4772. (21) Yamauchi, Y. J. Ceram. Soc. Jpn. 2013, 121 (1417), 831−840. (22) Wang, L.; Yamauchi, Y. J. Am. Chem. Soc. 2013, 135 (45), 16762−16765. (23) Ataee-Esfahani, H.; Nemoto, Y.; Wang, L.; Yamauchi, Y. Chem. Commun. 2011, 47 (13), 3885−3887. (24) Lee, H.; Habas, S. E.; Kweskin, S.; Butcher, D.; Somorjai, G. A.; Yang, P. Angew. Chem. 2006, 118 (46), 7988−7992. (25) Ren, J.; Shi, W.; Li, K.; Ma, Z. Sens. Actuators, B 2012, 163 (1), 115−120.
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DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
Facile Detection of Troponin I Using Dendritic Platinum Nanoparticles and Capillary Tube Indicators Sanghee Lee, Donghoon Kwon, Changyong Yim, and Sangmin Jeon* Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 790-784, Republic of Korea ABSTRACT: A facile method was developed for the detection of Troponin I (TnI) using dendritic platinum nanoparticles and capillary tube indicators. Dendritic platinum nanoparticles were functionalized with TnI antibodies, which were used to capture TnI in human serum. The captured TnI was conjugated to the inner surface of a glass vial, to which a hydrogen peroxide (H2O2) solution was added. After the glass vial was sealed with a screw cap containing a silicon septum, a capillary tube containing a drop of ink was inserted through the septum. The catalytic dissociation of H2O2 to water and oxygen increased the pressure inside the glass vial and raised the ink level in the capillary tube. The ink level increased with the platinum nanoparticle concentration, which is proportional to the TnI concentration. The sensitivity of this assay for TnI in human serum after a 5 min dissociation reaction, detected with the naked eye, was 0.1 ng/mL, which was better than the sensitivity of the conventional colorimetric method using the TMB oxidation reaction under the same experimental conditions. A control experiment using alpha-fetoprotein, interleukin-5, and C-reactive protein revealed that the developed method was highly selective for the detection of TnI. require bulky instrumentation. In contrast, the lateral flow immunoassay (LFA),10,11 which does not require any read-out instrumentation, has been used as a point-of-care platform; however, it has the critical disadvantage of low sensitivity (∼1 ng/mL). The sensitivity of the LFA could be improved by labeling with fluorescent or luminescent dyes, but this would require bulky and expensive detection systems.12,13 We demonstrate here a facile and sensitive method for measuring TnI concentrations using an alcohol thermometerlike glass vial and dendritic platinum nanoparticles. In this method, TnI in human serum was captured by antibodyfunctionalized dendritic platinum nanoparticles, and then conjugated to the inner surface of a glass vial functionalized with TnI antibodies. After addition of a hydrogen peroxide (H2O2) solution, the vial was sealed with a capillary tube containing a drop of ink. The pressure increase inside the glass vial caused by the catalytic dissociation of H2O2 to H2O and O2 increased the ink level in the capillary tube. This increase, detected by the naked eye, was used to determine the TnI concentration. The detection limit of the assay was found to be 0.1 ng/mL of TnI in human serum after a 5 min dissociation reaction.
L
iquid-in-glass thermometers, such as alcohol and mercury thermometers, are frequently encountered in our daily lives at home, in hospitals, and in school laboratories. In these devices, the volume of alcohol changes with temperature, and the resulting alcohol level is easily converted to temperature using an inscribed scale in the capillary tube. Although state-ofthe-art temperature measurement devices such as remote infrared detectors are available at reasonable prices, alcohol thermometers are still widely used owing to their many advantages, such as low cost, no need for external power, high reliability, and ease of use. The elegance of an alcohol thermometer lies mainly in its unique structure, consisting of a relatively large glass bulb containing red-colored alcohol and a narrow capillary tube connected to the glass bulb. This structure amplifies the small change in the alcohol volume inside a glass bulb, translating it to a large change in the alcohol level inside the capillary tube. This simple method for signal amplification may be utilized to measure biomarkers such as Troponin. Troponin is a complex of three subtype proteins: troponin I (TnI), troponin C, and troponin T. Troponin is considered a specific marker for myocardial damage because its concentration in blood increases upon damage to cardiac muscles. A number of immunoassays have been developed for measuring Troponin concentrations using microresonators,1,2 surface plasmon resonance (SPR),3−5 surface acoustic wave (SAW) sensors,6 and electrochemical sensors.7−9 However, these methods are not practical for most applications because they © 2015 American Chemical Society
Received: March 9, 2015 Accepted: April 20, 2015 Published: April 20, 2015 5004
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
Article
Analytical Chemistry
Figure 1. Schematic illustration of the detection of TnI using glass vials and capillary tubes with an ink indicator.
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Detection of TnI Using Decomposition of H2O2. Figure 1 shows a schematic illustration of the experimental procedure for the detection of TnI using an antibody-functionalized glass vial and a capillary tube containing a drop of red ink (safranin in an aqueous solution). First, 20 μL of a 2 mg/mL solution of antibody-functionalized platinum nanoparticles was added to 400 μL of TnI-spiked human serum. After incubation for 10 min at room temperature with gentle shaking, the sample containing TnI−Pt complexes was added to a gastight glass vial whose inner surfaces were functionalized with TnI antibodies. After incubation for 10 min at room temperature to allow TnI− Pt complexes to bind onto the glass surfaces, the glass vial was washed several times with 0.1% Tween 20 and PB. A 34.5% H2O2 solution was added to the sample, and the vial was sealed with a twist cap containing a silicon septum. A capillary tube containing a drop of ink was then inserted through the septum. The catalytic dissociation of H2O2 to H2O and O2 increased the pressure inside the glass vial, causing the ink level in the capillary tube to rise. The resulting rise was monitored with the naked eye or using a digital camera. To compare the sensitivity of the developed assay with the conventional colorimetric method based on the TMB oxidation reaction, we added TMB to the solution to induce a color change.
EXPERIMENTAL SECTION Materials. Chloroplatinic acid hexahydrate, 1 M sodium hydroxide solution, ascorbic acid, 3-aminopropyltriethoxysilane (APTES), glutaraldehyde, TCEP (tris(2-carboxyethyl)phosphine), Tween 20, 3,3′,5,5′-tetramethylbenzidine, and bovine serum albumin (BSA) were purchased from SigmaAldrich (St. Louis, MO, USA) and used as received without further treatment. TnI antigen and monoclonal antibodies against TnI (4T21 560 and 4T21 19C7) were purchased from Fitzgerald Industries (Acton, MA, USA) and Abcam HyTest Ltd. (Turku, Finland), respectively. Deionized water (18.3 MΩ· cm) was obtained using a reverse osmosis water system (Human Science, Korea) and used for making phosphate buffer (PB). Hydrochloric acid and H2O2 were purchased from Samchun (Pyeongtaek, Korea), and ethanol and methanol were purchased from Merck (Darmstadt, Germany). Glass vials (2 mL) and plastic screw caps with silicon septa were purchased from Agilent Technologies (Santa Clara, CA, USA). Preparation of the Capture-Antibody-Immobilized Glass Vial. A glass vial was cleaned by soaking in a mixture of hydrochloric acid and methanol for 1 h and then rinsed several times with methanol and water.14 After drying under a nitrogen flow, the glass vial was treated with 1% APTES in ethanol for 12 h to produce amine groups on the inner surface of the vial. A 1 mL, 4 μL/mL TnI antibody (4T21 560) solution was added to the glass vial, and the glass vial surface was functionalized with the antibodies using glutaraldehyde as a cross-linker. The glass vial surface was then blocked with a mixture of 1% BSA and 1% Tween 20 at 4 °C for 6 h to prevent nonspecific binding. Preparation of Detection-Antibody-Functionalized Platinum Nanoparticles. Dendritic platinum nanoparticles were synthesized by mixing 20 mL of 1.45 mM H2PtCl6, 250 μL of 1 M ascorbic acid, and 150 μL of 1 M sodium hydroxide.15 After the solution was heated at 60 °C for 10 min, the solution color changed from pale yellow to brown. The synthesized platinum nanoparticles were purified several times by centrifugation at 14000 rpm for 5 min. A half-fragment detection antibody (4T21 19C7)16 for easy immobilization of the antibody onto platinum nanoparticles with the desirable orientation was obtained by adding 10 μL of 5 mM TCEP in PB to 400 μL of PB containing 10 μg of detection antibody and incubating for 1 h at room temperature. After immobilization of the half-fragment antibody on platinum nanoparticles via Pt−S bonds, the functionalized nanoparticles were rinsed with PB solution several times, then 0.1% Tween 20 and sucrose were added to the solution to prevent nonspecific binding.
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RESULTS AND DISCUSSION Figure 2a,b show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images, respectively, of dendritic platinum nanoparticles. The average size of the dendritic nanoparticles was ∼40 nm, each of which consisted of tens of ∼5 nm platinum nanoparticles. The inset of Figure 2b shows the high-resolution TEM image of the dendritic platinum nanoparticles, indicating that the platinum consists of crystalline nanoparticles. Addition of the platinum nanoparticles to a H2O2 solution induces the dissociation of H2O2 molecules into H2O and O2. Natural enzymes such as catalase may be used for the dissociation of H2O2 molecules, but their activities are degraded over time; thus, they are not suitable for long-term storage.17,18 Because the catalytic activity of platinum nanoparticles is substantially affected by nanoparticle surface area, dendritic platinum nanoparticles were used in this experiment instead of single, large platinum nanoparticles.19−23 The X-ray diffraction (XRD) pattern for the platinum nanoparticles on a silicon wafer (Figure 2c) confirms that the dendritic platinum consists of crystalline nanoparticles. Figure 2d compares the changes in ink level during the catalytic dissociation of H2O2 caused by the dendritic platinum nanoparticles with that caused by conventional platinum nanoparticles synthesized by different methods.24,25 The results 5005
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
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Analytical Chemistry
Figure 2. (a) SEM and (b) TEM images of dendritic platinum nanoparticles. The average size of the platinum nanoparticles was ∼40 nm. The high-resolution TEM image in the inset of panel b indicates that the dendritic platinum consists of crystalline nanoparticles. (c) XRD pattern of synthesized dendritic platinum nanoparticles. (d) Time-dependent changes in ink level due to the dissociation of H2O2 by dendritic platinum nanoparticles (red circle) and conventional platinum nanoparticles (black square).
Figure 3. (a) Optical images of the ink level at various concentrations of TnI at 0, 1, 2, 3, 4, and 5 min after addition of H2O2 to the glass vial. The spacing between two adjacent lines is 5 mm. (b) Time-dependent changes in the ink level and the concentration of the produced O2 at various TnI concentrations: ■, 0 ng/mL; •, 0.1 ng/mL; ▲, 0.5 ng/ mL; ▼, 1 ng/mL; ◀, 10 ng/mL; ▶, 100 ng/mL. The right y-axis shows the molar concentration of O2 produced by the dissociation of H2O2.
showed that dendritic platinum nanoparticles induced far larger increases in the ink level than conventional platinum nanoparticles, despite the fact that the number, concentration, and size of the particles were nearly identical. Figure 3a shows optical images of the time-dependent changes in ink level as a function of TnI concentration. The spacing between two adjacent lines is 5 mm, and each snapshot was obtained at 0, 1, 2, 3, 4, and 5 min after the addition of H2O2 to the glass vial containing TnI−Pt complexes. Negligible changes were observed for 0 ng/mL TnI, indicating that platinum nanoparticles did not bind nonspecifically to the glass vial surface. The level of ink increased with increases in TnI concentration and reaction time, and the change in ink level could be clearly detected with the naked eye. The change in ink level for the 100 ng/mL TnI solution after a 5 min reaction was 10.6 mm, which was 8 times larger than that for a 0.1 ng/mL TnI solution. Figure 3b shows the time-dependent changes in ink level and the molar concentration of O2 produced as a function of TnI concentration. The change in level can be converted to the volume change by simply multiplying the inner diameter of the capillary tube (1.8 mm) by the change in level. Assuming that the volume change is induced solely by the O2 produced by catalytic dissociation of H2O2, changes in the ink level can be converted into the molar concentrations of the produced O2 (Figure 3b, right y-axis). The level of ink (i.e., the O2 concentration) increased almost linearly with reaction time, indicating that the catalytic production of O2 by platinum nanoparticles is a zero-order reaction, because the rates are independent of substrate concentration (i.e., H2O2 concentration). Zero-order reactions are typically observed for enzyme-catalyzed reactions in which the concentration of enzyme (i.e., platinum nanoparticle) controls the reaction rate. In zero-order kinetics, the reaction rate is dependent on the enzyme concentration, and the derivative of the product
concentration as a function of time (i.e., the slopes of the linear lines fitting the data) corresponds to the rate constant. Figure 4 shows changes in the ink level as a function of TnI concentration in serum after the catalytic dissociation of H2O2
Figure 4. Changes in ink level as a function of TnI concentration after the catalytic dissociation of H2O2 for 5 min.
for 5 min. The ink level increased linearly with the log of TnI concentration. Based on the three-sigma method, the detection limit of the assay with the naked eye was found to be 0.1 ng/ mL of TnI in human serum after a 5 min catalytic dissociation reaction, a limit suitable for the diagnosis of cardiac problems. Because the ink level increases with reaction time, the detection limit could be improved further by increasing the reaction time. A series of control experiments was conducted to examine the selectivity of the assay. After TnI antibody-functionalized 5006
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
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Figure 5. (a) Changes in ink level measured for human serum spiked with various proteins at 100 ng/mL. (b) Optical images of cuvettes containing different concentrations of TnI after a 5 min TMB oxidation reaction. (c) Changes in absorbance of the solutions as a function of TnI concentration.
Notes
dendritic platinum nanoparticles were dispersed in human serum spiked with 100 ng/mL α-fetoprotein (AFP), interlukin5 (IL-5) or C-reactive protein (CRP), they were allowed to bind onto the glass vial surface functionalized with anti-TnI antibodies. Figure 5a shows that changes in the ink level for AFP, IL-5, and CRP after the catalytic dissociation of H2O2 for 5 min were far smaller than that for 100 ng/mL TnI, indicating that the detection of TnI in human serum was highly selective. In addition, the sensitivity of the developed assay was compared with that of the conventional TMB oxidation reaction by platinum nanoparticles. Figure 5b shows optical images of cuvettes containing different concentrations of TnI after a 5 min TMB oxidation reaction. The color of the solution became blue as the concentration of TnI increased owing to the catalytic oxidation of TMB to tetramethylbenzidine diimine by platinum nanoparticles in the presence of H2O2. The resulting production of tetramethylbenzidine diimine causes the solution to turn blue. The detection limit of the assay with the naked eye was 10 ng/mL of TnI in human serum, a limit 2 orders of magnitude lower than that of our method under the same experimental conditions. The concentration of TnI was also determined quantitatively using a UV−vis spectrometer by measuring absorbance at 370 nm. Figure 5c shows the changes in absorbance as a function of the concentration of TnI. The detection limit with the spectrometer was found to be 1 ng/mL, still lower than that of our method with the naked eye. The higher sensitivity of our assay is attributable to the production of bubbles instead of a chemical product. Because the gas volume is 1000 times larger than the liquid volume with the same number of molecules, the volume change due to the production of hydrogen is much greater than the color change due to the production of tetramethylbenzidine diimine.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by a National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2014R1A2A2A01007027).
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CONCLUSION In summary, we have developed a facile method for the detection of TnI in human serum using antibody-functionalized dendritic platinum nanoparticles and a capillary tube containing a drop of ink. The pressure increase caused by the catalytic dissociation of H2O2 by platinum nanoparticles induces an increase in ink level, which is easily identified with the naked eye. The detection limit of the assay was 0.1 ng/mL TnI in human serum after a 5 min catalytic reaction. Because the ink level increases further with reaction time, the limit of detection could be improved by increasing the reaction time. This simple and cost-effective method may be especially beneficial for people in underdeveloped countries.
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*S. Jeon. E-mail: [email protected]. 5007
DOI: 10.1021/acs.analchem.5b00921 Anal. Chem. 2015, 87, 5004−5008
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