Article pubs.acs.org/ac
Black Phosphorus Nanoparticle Labels for Immunoassays via Hydrogen Evolution Reaction Mediation Carmen C. Mayorga-Martinez,† Naziah Mohamad Latiff,† Alex Yong Sheng Eng,† Zdeněk Sofer,‡ and Martin Pumera*,† †
Division of Chemistry & Biological Chemistry, School of Physical Mathematical Science, Nanyang Technological University, Singapore 637371, Singapore ‡ Department of Inorganic Chemistry, University of Chemistry and Technology Prague, Technicka 5, 166 28, Prague 6, Czech Republic ABSTRACT: Black phosphorus is an emerging layered material. Its nanoparticles show an increased bandgap when compared to bulk materials and they are typically fabricated by ultrasonication of macroscopic black phosphorus crystals. Here we fabricate black phosphorus nanoparticles (BP NPs) by solution based electrochemical exfoliation with bipolar electrodes, which induces opposite potentials on the opposite ends of black phosphorus macroparticles thereby leading to its decomposition into nanoparticles. BP NPs have enhanced catalytic effect on the hydrogen evolution reaction (HER) relative to black phosphorus macroparticles. We utilize black phosphorus nanoparticles as electrocatalytic tags in a competitive immunoassay for rabbit immunoglobulin G (IgG) detection. The detection signal is produced via nanoimpacts of the BP NPs followed by HER catalysis.
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ayered and 2D materials have been used for several sensing and biosensing applications.1,2 Recently, layered black phosphorus has received significant amount of attention as it can be exfoliated, similarly to graphite to its single layer 2D material.3−7 Black phosphorus is an elemental material with strong electrical, magnetic, and electrochemical anisotropies with low toxicity.8−10 This material has been suggested to play a key role in many devices spanning across broad applications ranging from gas sensing11−14 to nanoelectronics15−17 and has more recently expanded into catalysis.18 Its electronic and optical properties are further known to change with a decreasing number of layers.19,20 Black phosphorus nanoparticles (BP NPs) or quantum dots also demonstrate altered properties when compared to its bulk form.4,17,19 BP NPs are typically prepared by mechanical decomposition of millimeter or micrometer sized grains of black phosphorus with subsequent ultrasonications.4,17 There is immense potential in the use of these nanoparticles, in a similar manner to other nanoparticles of gold or carbon, as labels for sensors and biosensors. Herein, for the first time, we report a single-step solution based electrochemical exfoliation with bipolar electrodes for exfoliation and downsizing of the layered black phosphorus microparticles to nanoparticles with enhanced electrocatalytic activity for the hydrogen evolution reaction. We show that we can utilize these BP NPs for protein detection using impact electrochemistry21,22 via hydrogen evolution mediation. The BP NPs label system of a magnetoimmunoassay based on impact electrochemistry detection presents a new concept in biosensing technology. © XXXX American Chemical Society
EXPERIMENTAL SECTION
Structural and Morphological Characterization. Phoibos 100 spectrometer with monochromatic Mg X-ray radiation source (SPECS, Germany) and UV−vis spectrometer (Shimadzu UV-2500) were used for perform the X-ray photoelectron and UV−vis spectroscopy, respectively. JEOL 7600F field-emission scanning electron microscope (JEOL, Japan) in TED mode at accelerating voltage of 30 kV was used to obtain the scanning transmission electron micrographs. For the dynamic light scattering experiments, PMMA cuvettes and Zetasizer Nano ZS (Malvern Instruments, England) were used and the measurements were done at 20 °C. Synthesis of BP NPs. First, black phosphorus crystal was prepared by a reported protocol.13 In this way, 500 mg of Au/Sn alloy was obtained by melting stoichiometric amounts gold (99.99% purchased from Safina, Czech Republic) and tin (99.999% from STREM, Germany) in chloroform (99.9% from PENTA, Czech Republic), the reaction was performed under high vacuum conditions into a glass ampule. Then, 720 mg of red phosphorus (99.999% from Sigma-Aldrich, Czech Republic) and 15 mg of SnI4 (99.8% from PENTA, Czech Republic) were added to the reaction and the ampule was sealed by using an oxygen/hydrogen torch. The synthesis of SnI4 was performed using iodine (99.8% from PENTA, Czech Republic) Received: June 23, 2016 Accepted: September 23, 2016
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DOI: 10.1021/acs.analchem.6b02422 Anal. Chem. XXXX, XXX, XXX−XXX
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potential of 0.01 V (root-mean-square, rms) plus −0.81 V vs RHE polarization potential in a frequency range of 1 mHz to 100 kHz. The overpotential for HER was measured at −10 mA cm−2 current density. The electrochemical detection of BP NPs captured through magneto immunoassay was performed via HER by spike count of the chronoamperometry scan. The threecomponent screen printed electrode (obtained from eDAQ Instruments) with a magnet disc attached on the reverse side of the working electrode area was positioned in a horizontal manner; a fixed 15 μL of the magnetic bead/antirabbit IgG/ BPNPs-rabbit IgG complex containing 0, 2, 10, 50, 100 ng/mL of free IgG (prepared previously) was drop-casted onto the working electrode area. After 1 min, 15 μL of 1 M H2SO4 was drop-casted, and with the help of a micropipet was gently mixed and distributed onto the three-component electrode. A reductive potential of −0.88 V vs RHE was applied for 100 s, and chronoamperometry scans were carried out immediately thereafter to detect all possible impact of the particles onto the electrode.
and tin in chloroform under reflux, followed by the purification of SnI4 and recrystallization from chloroform. Next, the reaction was heated at 400 °C for 3 h using a muffle furnace. Afterwards, the temperature of the reaction was increased to 600 °C and held for 24 h followed by the furnace cooling (overnight) until it reached room temperature. For removal of white phosphorus formed as a side product, the obtained black phosphorus platelets (∼5 mm × 2 mm) were washed with CS2. Subsequently, the BP crystals (0.5 mg/mL) obtained were sonicated for 4 h for pulverization to microparticles and Na2SO4 was added to the solution to reach a final concentration of 0.5 M. The resulted solution was transferred to the two-platinum electrodes electrochemical system. The distance between the platinum electrodes was 2 cm and 10 V DC potential was applied for 30 min. The obtained solution was left to stand for 1 week, and the supernatants were separated from the pellet. For the structural characterization (XPS and STEM), the solution was desalinated using dialysis membrane (cutoff below 1 kDa). Competitive Magneto-Immuno Assay Preparation. The 3 mg/mL magnetic beads tosyl-activated (Dynabeads M280 from Invitrogen, Singapore) solution was washed twice in 0.1 M sodium borate buffer pH 9.2. The borate buffer was prepared using boric acid 99.5% purchased from Sigma-Aldrich, Singapore. Then, 40 μg/mL polyclonal antirabbit IgG produced in goat (from Sigma-Aldrich, Singapore) solution was added and, the mixture was incubated overnight at 37 °C (400 rpm). The resulted conjugated (MB/antirabbit IgG) was washed with tween buffer. The tween buffer was prepared using 0.01 M PBS buffer, pH 7.4 (the PBS buffer was prepared using a tablet from Sigma-Aldrich, Singapore). After that, the blocking step was performed by incubation for 1 h at 25 °C (400 rpm) of MB/ antirabbit IgG complex in 5% BSA-PBS buffer. The resulting solution was washed with PBS buffer pH 7.4. In parallel, IgG from rabbit serum (1 μg/mL) was incubated with BP NPs during 1 h at 25 °C and 400 rpm. The pH of the BP NPs solution was previously adjusted using PBS buffer pH 7.4. Once the rabbit IgG/BP NPs was obtained, the conjugate was blocked by adding 15 μL of a 5% BSA solution prepared in Milli-Q water and incubating for 20 min at 650 rpm and 25 °C. For removal excess BP NPs, the resulted solution was centrifuged for 2 h at 4 °C (14 000 rpm). The supernatant was removed, and the pellet was resuspended using 80 μL of PBS buffer pH 7.4. Finally, the MB/ antirabbit IgG and rabbit IgG labeled with BP NPs were incubated (for 1 h under 400 rpm agitation at 25 °C) in the presence of the desired concentration of free rabbit IgG. In this way, 80 μL of magnetic bead/antirabbit IgG complex was separated from the supernatant and mixed with 80 μL of BP NPsrabbit IgG (previously conjugated) containing 0, 2, 10, 50, and 100 ng/mL of rabbit IgG. Once the conjugated species was obtained, the washing step was performed using 0.5% tween-PBS buffer, PBS buffer, and Milli-Q water. Electrochemical Measurement. The electrochemical measurements were performed using Autolab PGSTAT204/ FRA32 M (Eco Chemie, Utrecht, The Netherlands). An electrochemical cell composed of glassy carbon (GC) electrode as the working electrode, Pt counter electrode, and an Ag/AgCl reference electrode were used. The efficiency of BP NPs for the hydrogen evolution reaction was evaluated using linear sweep voltammetry (at a scan rate of 2 mV/s) and electrochemical impedance spectroscopy (EIS). For this aim, the GC working electrode was modified with 3 μL of BP NPs solution by dropcasting. In all the cases, the measurements were done in 0.5 M H2SO4. EIS measurements were performed using an AC
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RESULTS AND DISCUSSION In this study, we describe the utilization of BP NPs as labels for immunoassays using impact electrochemistry as a mode of Scheme 1. Synthesis of Black Phosphorus Nanoparticles (BP NP) by Solution-based Electrochemical Exfoliation with Bipolar Electrodes
detection mediated by hydrogen evolution reaction on BP nanoparticles. There have been many research efforts in developing efficient methods for downsizing and exfoliating layered black phosphorus crystals to BP NPs and phosphorene, all of which were based on mechanical forces, such as ultrasonication.4−7,17−19,23 We introduce a new method for fabrication of such nanoparticles using the solution based electrochemical exfoliation with bipolar electrodes.24 To fabricate BP NPs, we first synthesized large BP crystals.13 The exfoliation and downsizing method, consists of applying a constant DC potential of 10 V between two platinum electrodes, see Scheme 1. Potential difference is induced on the BP particles at opposite sides of the particle,23 which leads to fragmentation of the BP particles into nanoparticles. B
DOI: 10.1021/acs.analchem.6b02422 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Photos of black phosphorus macroparticles (BP MPs) solution before (A) and after (B) solution based electrochemical exfoliation with bipolar electrodes process. STEM micrographs of black phosphorus nanoparticles (BP NPs) (C).
Figure 2. Particle size distribution measured by DLS (A) and UV−vis absorption spectra (B) of black phosphorus nanoparticles (BP NPs). Highresolution XPS spectra of black phosphorus macroparticles (BP MPs) and BP NPs (C).
BP NPs for hydrogen evolution reaction (Figure 3A). The overpotential for HER at BP NPs was −0.81 V vs the reversible hydrogen electrode (RHE), whereas the overpotential for BP macroparticles was seen at −1.24 V vs RHE. The polarization curve for bare GC (glassy carbon) produced an overpotential at −0.97 V vs RHE. These results clearly demonstrate higher electrocatalytic activity of BP NPs compared to BP macroparticles and GC electrode. EIS measurements show the enhanced catalytic activity of BP NPs by the electrode kinetics under HER operating conditions. Figure 3B shows a dramatic decrease in charge transfer resistance (Rct) for the BP NPs (1.39 kΩ) relative to the BP macroparticles (37.6 kΩ). Accordingly, the EIS results show that BP NPs exhibit lower electron transfer resistance when compared to the BP macroparticles. The exfoliation process exposed more catalytic edges sites8 of the BP NPs (see Figure 3B). These factors contributed to the enhanced catalytic performance of HER observed. Subsequently, the competitive magneto immunoassay for protein detection using BP NPs as a label was performed. Scheme 2 (not to scale) presents the depiction of the complete assay. First, tosyl-activated paramagnetic beads (MB) were conjugated with antirabbit IgG (a). In parallel, the rabbit IgG is labeled with BP NPs (a′) and this complex was then conjugated
Figure 1 shows the BP macroparticles solutions before (A) and after (B) the exfoliation process. We can clearly see that the exfoliation process occurs with the disappearance of the characteristic dark color of the BP macroparticle solution. The BP NPs suspension was settled after exfoliation process for a week and the clear solution was separated from the pellet. A dialysis membrane with a cutoff below 1 kDa was used for desalination of the BP NPs solution. Scanning transmission electron microscopy (STEM) was performed on the dialyzed solution as displayed in Figure 1C, which shows the presence of platelet-like particles of various sizes. The hydrodynamic radii of the BP NPs were around 70 nm from the data of the dynamic light scattering (DLS) (Figure 2A). For evaluation of the structural and chemical changes of the resulted BP NPs, UV−visible spectroscopy (Figure 2B) and Xray photoelectron spectroscopy (XPS) (Figure 2C) were performed. The optical absorption spectra of BP NPs show characteristic absorption bands at UV and NIR regions; similar results were reported before in the literature.17,23 The characteristic phosphorus and surface oxidation features17,23,25 were observed in the high-resolution XPS spectra of BP crystals and BP NPs, see Figure 2C. Linear scan voltammetry (LVS) measurements were carried out for evaluating the performance of the BP macroparticles and C
DOI: 10.1021/acs.analchem.6b02422 Anal. Chem. XXXX, XXX, XXX−XXX
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more negative than the overpotential of HER for BP NPs (see previous section) to perform the chronoamperometry (Figure 4A).27 When the MBs/antirabbit IgG/rabbit IgG-BP NPs are in contact with H2SO4, the BP NPs are freed and thereby strike the electrode surface, which generates a current response (reductive spike) that corresponds to a proton reduction (see Figure 4A, red line). When the concentration of free rabbit IgG increases, the number of the spikes decrease as expected in a competitive immunoassay. This is because the amount of the BP NPs decreases in the magneto-immunoassay as the immunoassay is based on competitive binding between rabbit IgG-BP NP and rabbit IgG. The number of the spikes for the magnetoimmunoassay complex at different concentrations of free rabbit IgG are larger than the blank control (Figure 4A, black line). To construct a calibration curve for rabbit IgG detection, we utilized the number of spikes in the first 40 s of chronoamperograms (Figure 4B). Only spikes with at least twice the amplitude of the background signal were considered as impact signals from BP NP.27 The curve relating the number of spikes as a function of rabbit IgG concentration shows that the spike count decreases as protein concentration increases (Figure 4B), as expected from competitive immunoassay. Furthermore, a calibration curve relating the inverse of number of spikes vs rabbit IgG concentration (see Figure 4C) shows a linear range from 2 to 100 ng/mL with an R of 0.9429. Moreover, the system shows a low limit of detection (LOD) of 0.98 ng/mL. The reproducibility of the assay was evaluated getting a relative standard deviation (RSD) around 18% which indicates a good reproducibility of the system. Reproducibility experiments are performed according to the Bioanalytical Method Validation from Guidance for Industry of the Food and Drug Administration (FDA) federal agency of the United States.34 The RSD reported here represents the average of four different free rabbit IgG concentrations, with three replicates of the assay for each concentration. For the selectivity evaluation, immunoassays were performed in the absence of BP NPs and nonspecific protein (human hemoglobin) used instead of rabbit IgG. The small spike count in the control immunoassays observed demonstrated the high selectivity of the system (Figure 5B).
Figure 3. Linear scan voltammograms (A) and Nyquist plots (B) of black phosphorus macroparticles (BP MPs) and black phosphorus nanoparticles (BP NPs). Conditions: 0.5 M H2SO4, glassy carbon (GC) electrode was modified with 1.5 μg of materials by drop casting.
with MBs/antirabbit IgG in the presence of free rabbit IgG at the desired concentration to perform the competitive magneto immunoassay (b). Once the complex MBs/antirabbit IgG/rabbit IgG-BP NPs is formed, 15 μL of this solution was drop casted onto the screen-printed electrode followed by addition of 15 μl of 1 M H2SO4 (c). The acid leads to denaturation of the protein complex and release of BP NPs which consequently impacts the surface of the electrode (d). The detection of BP NPs (and thus of IgG concentration) was performed by the electrocatalysis of impacting BP NPs (nanoimpact method)4,20,21,25,26 via hydrogen evolution reaction (proton reduction) (e). Nanoimpact electrochemistry is a method that allows for determination of nanoparticle size, type, and concentration via direct oxidation or reduction of the nanoparticles or via catalysis of an electrochemical reaction (i.e., proton reduction) by particle impacting on the electrode surface.21,22,26−33 This method was used recently for DNA analysis, demonstrating their promising usage in the biosensing field.26 One can directly detect black phosphorus NPs by their oxidation from P0 to P5+ using impact electrochemistry.4 Here, we demonstrate the nanoimpact electrochemistry as a detection technique for protein quantification through HER by impact of BP NPs in sulfuric acid. The acid solution serves not only as an electrolyte but also functions as a denaturating agent which liberates the BP NPs. Consequently, the BP NPs are detected via catalysis of H+ reduction to H2 upon impact of BP NPs onto the electrode surface. Number of spikes is related to the number of attached black phosphorus nanoparticles which is in turn related to amount of analyte. The analytical protocol is based on catalytic events (spikes) which are catalyzed by black phosphorus nanoparticles. In order to perform detection of liberated BP NP via mediation of hydrogen evolution reaction, we have set a reduction potential of −0.88 V (vs RHE), which is slightly
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CONCLUSION Here we implemented a conceptually new approach of fabricating black phosphorus nanoparticles that consist in solution based electrochemical exfoliation with bipolar electrodes, yielding nanoparticles of 40−200 nm in size with a maximum at ∼70 nm. Black phosphorus nanoparticles showed hydrogen evolution activity at more positive potentials than black phosphorus macroparticles. We demonstrated the utility of these black phosphorus nanoparticles as a label for protein detection in a competitive immunoassay through hydrogen evolution reaction using an optimized nanoimpact method of black phosphorus NPs anchored to magnetic beads through immunoassay for the quantification of rabbit IgG. This developed system shows competitive analytical performance compared with the systems reported for gold nanoparticles. The concept of employing black phosphorus nanoparticles on biomolecular labels is very attractive due to low toxicity of black phosphorus, thus it is expected to pave its way in the development of cost efficient biosensors for different target detection. D
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Scheme 2. Schematic of the Competitive Magneto-Immunoassay for Protein Using BP NPs as a Tag and HER Electrocatalysis (Proton Reduction) By Impact Electrochemistry (Spikes Count) as a Detection Techniquea
a
(a and a′) Magnetic beads (MB) conjugation with anti-rabbit IgG and tag-labelled step of rabbit IgG with BP NPs, respectively, (b) incubation of the MB/anti-rabbit IgG conjugate with rabbit IgG/BP NPs in the presence of different concentrations of free rabbit IgG. Electrochemical detection by nano-impact method: (c) MB-based complex drop casted onto the working electrode in the presence of 0.5 M of H2SO4 and detection (e) after impact of the liberated BP NPs (d).
Figure 4. Chronoamperograms of the competitive magneto-immunoassay at different rabbit IgG concentrations in 0.5 M H2SO4 at −0.88 V vs RHE (A). Curve of the spike count (B) and the inverse of spikes counts (C) as a function of different rabbit IgG concentrations.
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Figure 5. Selectivity evaluation: enlarged chronoamperograms (A) and summary of the spike count (B) obtained for the magneto-immunoassay performed in the presence and absence of BP NPs and in the presence of human hemoglobin.
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AUTHOR INFORMATION
Corresponding Author
*Fax: (+65) 6791 1961. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS M.P. was supported by Tier 2 grant (Grant MOE2013-T2-1-056; ARC 35/13) from the Ministry of Education, Singapore. Z.S. was supported by the Czech Science Foundation (GACR Grant No. 15-09001S).
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