Materials
Potassium permanganate (KMnO4, ≥ 99%), citric acid (≥ 99.5%), hydrochloric acid (HCl, 37%), copper(II) sulfate pentahydrate (≥ 98%), laccase from Trametes versicolor (≥ 0.5 U/mg), bovine serum albumin (BSA, ≥ 96%), horseradish peroxidase (HRP, ≥ 250 U/mg), phosphate buffered saline (PBS), 2-(N-morpholino)ethanesulfonic acid (MES, ≥ 99%), 3-aminopropyl triethoxysilane (APTES, 99%), 2,4-dichlorophenol (2,4-DP, ≥ 98%), 4-aminoantipyrine (4-AP, ≥ 97%), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, ≥ 98%), hydrogen peroxide (H2O2, 30% aqueous solution), dopamine hydrochloride (≥ 98%), epinephrine (≥ 99%), phenol (99.0-100.5%), bisphenol A (≥ 99%), hydroquinone (≥ 99%), catechol (≥ 95%), 2-naphthol (≥ 99%), crystal violet (≥ 96%), neutral red (≥ 90%), and rhodamine B (≥ 95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water purified using a Milli-Q Purification System (Millipore, Darmstadt, Germany) was used to prepare all solutions. All chemicals were of analytical grade or higher and used as received without further purification.
Synthesis and characterization of MnO2 NFs, amine-functionalized MnO2 NFs, and H–Mn–Cu NFs
MnO2 NFs were synthesized according to a previous study, with some modifications [23]. Briefly, KMnO4 (80 mg) was dissolved in an aqueous HCl solution (1 M, 40 mL). Then, an aqueous citric acid solution (100 mM, 1 mL) was added to the solution and stirred for 30 min at room temperature (RT, 22 °C), yielding a color change from red violet to brown. The resulting MnO2 NFs were collected via centrifugation at 10,000 rpm for 5 min, washed with distilled water, and dried at 50 °C under vacuum. Amine-functionalized MnO2 NFs were synthesized by dispersing MnO2 NFs (150 mg) into a mixture of distilled water (2 mL) and absolute ethanol (300 mL), followed by sonication for 10 min. APTES (0.6 mL) was added to the mixture under constant stirring for 7 h. The resulting amine-functionalized MnO2 NFs were collected by centrifugation at 10,000 rpm for 5 min, washed with ethanol, and dried at 50 °C under vacuum for 1 d. H–Mn–Cu NFs were synthesized according to a previously reported self-assembly method, with marginal modifications [24]. Typically, 60 µL of aqueous CuSO4 solution (120 mM) was added to 9 mL of PBS (10 mM, pH 7.4) containing the amine-functionalized MnO2 NFs (0.1 mg mL− 1), followed by three days of incubation at RT. The resulting H–Mn–Cu NFs were then collected using centrifugation at 10,000 rpm for 5 min, washed three times with deionized water, and dried at 50 °C under vacuum. As a control, Cu3(PO4)2 precipitates were prepared by incubating a CuSO4 solution in PBS for three days at RT, as previously reported [25].
The size, morphology, and elemental composition of the synthesized nanoflowers were analyzed using scanning electron microscopy (SEM) (Magellan 400 microscope; FEI Co., Cambridge, UK) with an energy-dispersive X-ray spectrometer (EDS; Bruker, Billerica, MA). For SEM, a suspension of nanoflowers was dropped on a silicon wafer and dried overnight at RT. Fourier transform infrared (FT-IR) spectra and X-ray diffraction (XRD) patterns of the MnO2 NFs, amine-functionalized MnO2 NFs, H–Mn–Cu NFs, and Cu3(PO4)2 precipitates prepared by incubating only copper sulfate in PBS without MnO2 NFs were obtained using an FT-IR spectrophotometer (FT/IR-4600; JASCO, Easton, MD) and an X-ray diffractometer (D/MAX-2500; Rigaku Corporation, Tokyo, Japan), respectively. The specific surface area, pore diameter distribution, and pore volume were obtained from N2 physisorption isotherms obtained with a physisorption analyzer (3Flex; Micromeritics, GA, USA) using the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods. X-ray photoelectron spectroscopy (XPS) (Sigma Probe, Thermo Scientific, WI, USA) was performed to investigate the electronic states of the Mn and Cu within the H-Mn-Cu NFs. The elemental ratio between Mn and Cu within the H-Mn-Cu NFs was determined via an inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7700 S, CA, USA) analysis.
Determination of laccase-mimicking activity of H–Mn–Cu NFs
Laccase-like activity was measured using the chromogenic reaction of phenolic compounds with 4-AP as follows: First, 2,4-DP (1 mg mL1, 100 µL) was mixed with 4-AP (1 mg mL− 1, 100 µL) in MES buffer (50 mM, pH 6.8, 700 µL). Free laccase or H–Mn–Cu NFs (1 mg mL− 1, 100 µL) were then added. After reacting for 40 min at RT, the mixture was centrifuged at 10,000 rpm for 2 min, and the absorbance of the supernatant was recorded in scanning mode or at 510 nm using a microplate reader (Synergy H1; BioTek, VT, USA, at the Core-facility for Bionano Materials in Gachon University). Other phenolic substrates (phenol, bisphenol A, hydroquinone, catechol, 2-naphthol, and dopamine) were used as target compounds instead of 2,4-DP; the other assay procedures were the same as those described above.
The effects of pH on the laccase-like activity of H–Mn–Cu NFs were examined following the same procedures but using MES buffer solutions prepared from pH 3 to 10. The effects of incubation temperature on the activity of H–Mn–Cu NFs were also explored following the same procedures but incubated under diverse temperature conditions (4–80 °C). The relative activity (%) was calculated using the ratio of measured activity to the standard activity, measured at pH 6.8 and RT. Stabilities for pH, temperature, and ionic strength of H–Mn–Cu NFs and free laccase were evaluated by incubating them in aqueous buffer (MES, 50 mM) at different pH values (pH 3–10) for 5 h, different temperatures (4–80 °C) for 3 h, and different NaCl concentrations (0, 62.5, 125, 250, and 500 mM) for 10 h, followed by measurement of the residual activities using standard assay methods. The long-term operational stabilities of H–Mn–Cu NFs and free laccase were measured by assessing their daily activities during their incubation at RT under mild shaking conditions. The relative activity (%) was calculated as the ratio of residual activity to the initial activity of each sample.
Steady-state kinetic parameters were evaluated by performing the laccase-mediated reaction at RT in a 1.5-mL tube with H–Mn–Cu NFs or free laccase (both at concentrations of 0.1 mg mL− 1) in MES buffer (50 mM, pH 6.8). Epinephrine at various concentrations (9.4, 18.7, 37.5, 75, 150, 300, and 600 µM) was added to 1 mL of reaction buffer. After the substrates were mixed, the color changes were monitored in kinetic mode at 485 nm. The kinetic parameters were calculated based on the Michaelis–Menten equation: ν = Vmax × [S] / (Km + [S]), where ν is the initial velocity, Vmax is the maximal velocity, [S] is the concentration of the substrate, and Km is the Michaelis constant.
To evaluate the dopamine detection sensitivity of the H–Mn–Cu NFs, dopamine at various concentrations (100 µL) was mixed with 4-AP (1 mg mL− 1, 100 µL) and H–Mn–Cu NFs (1 mg mL− 1, 100 µL) in MES buffer (50 mM, pH 6.8, 700 µL), followed by incubation for 40 min at RT. After the reaction, the mixture was centrifuged at 10,000 rpm for 2 min, and the absorbance of the supernatant was recorded at 510 nm. To measure the detection sensitivity for epinephrine, epinephrine at diverse concentrations (100 µL) was mixed with H–Mn–Cu NFs (1 mg mL− 1, 100 µL) in MES buffer (50 mM, pH 6.8, 800 µL). The other procedures were the same as those described for the detection of dopamine, except that the absorbance at 485 nm, which corresponds to the oxidized epinephrine, was measured rather than 510 nm. The limit of detection (LOD) values were calculated according to the equation LOD = 3 S / K, where S is the standard deviation of the blank absorbance signals, and K is the slope of the calibration plot.
Degradation of dyes by H–Mn–Cu NFs or free laccase
The dye degradation efficiencies of H–Mn–Cu NFs and free laccase were assessed using crystal violet (CV), neutral red (NR), and rhodamine B (RB) as model dyes. First, H–Mn–Cu NFs or laccase (1 mg mL− 1, 1 mL) was mixed with the dye solution [9 mL at concentrations of 2.5 mg mL− 1 (CV), 7.5 mg mL− 1 (NR), or 1.5 mg mL− 1 (RB)]. The mixture was incubated in the dark with gentle shaking at RT. The dye degradation efficiencies of CV, NR, and RB were analyzed by measuring the absorption intensities at 590, 523, and 543 nm, respectively, at predetermined time points, using a microplate reader. Matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF, Bruker autoflex maX, Bruker Daltonics, MA, USA) mass spectrometry was performed to confirm the degradation of CV, NR, and RB by the incubation with H-Mn-Cu NFs.
H–Mn–Cu NFs-embedded paper microfluidic devices for colorimetric determination of phenolic neurotransmitters
Paper microfluidic devices, including H–Mn–Cu NFs, were constructed using a wax printing method [26]. The pattern was first designed using AutoCAD 2018, followed by printing wax on Whatman chromatography paper (grade 1) with a wax printer (ColorQube 8570DN; Xerox, Japan). The printed paper was placed on a hot plate at 170ºC for 2 min to melt the wax and then cooled at RT to form hydrophobic barriers.
For the detection of both dopamine and epinephrine on a single device, the microfluidic device was divided into two parts for the detection of dopamine (D) and epinephrine (E). In each part, there were three circular detection zones (6 mm in diameter), microfluidic channels (3 mm in width, and 6 mm in length), one half-circular sample zone (7.5 mm in diameter) for sample injection, and one circular control zone (6 mm in diameter). On the dopamine-detecting part, both H–Mn–Cu NFs and 4-AP were immobilized in the control zone, whereas only H–Mn–Cu NFs were immobilized in the control zone on the epinephrine-detecting part. To detect single neurotransmitters of either dopamine or epinephrine, the device was not divided and consisted of six circular detection zones and one circular sample zone connected to the microfluidic channels. For the dopamine-detecting device, two circular control zones (6 mm in diameter) were prepared, where the first contained both H–Mn–Cu NFs and 4-AP, and the other contained only 4-AP without the nanoflowers. For the epinephrine-detecting device, a circular control zone (6 mm in diameter) was prepared, where only the H–Mn–Cu NFs were immobilized.
To construct paper microfluid devices with incorporated H–Mn–Cu NFs, H–Mn–Cu NFs (10 mg mL− 1, 2 µL) were dropped onto the detection and control zones of the devices. 4-AP (5 mg mL− 1, 2 µL) was consecutively dropped on the dopamine detection zones and control zones. The paper device was then dried at 50 °C for 5 min. To detect phenolic neurotransmitters, 20 µL of the sample solution containing dopamine or epinephrine was dropped twice onto both parts of the half-circular sample zone or 40 µL of sample solution was dropped once onto the circular sample zone to detect single phenolic neurotransmitters of either dopamine or epinephrine. After 10 min, the resulting devices were directly used to obtain images with a smartphone (Galaxy S8 NOTE; Samsung, Korea), followed by conversion to a yellow scale, which was subjected to quantitative image processing using the ImageJ software (NIH).