Scheme 1 (a) Schematic illustration of the enzyme-immobilized
alginate microfibers and (b) colorimetric detection of glucose by
microfibers.
Materials and methods
2.1 Materials
Sodium alginate, calcium chloride (dihydrous) and
poly (acrylic acid) (PAA) solution
were purchased from Sigma-Aldrich (USA).
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl)
and N-hydroxy succinimide (NHS) were purchased from Aladdin Chemistry
(China). Glucose oxidase (GOX) from Aspergillus niger was
obtained from Solarbio (China).
Horseradish
peroxidase (HRP), 3,3’,5,5’-tetrame-thylbenzidine (TMB), and dimethyl
sulfoxide (DMSO) were purchased from Sangon Biotech (China). Bovine
serum albumin (BSA) was obtained from Beyotime (China).
Fluorescein-5-isothiocyanate (FITC) was purchased from Thermo Fisher
Scientific (USA). Triethylamine was obtained from Shanghai Lingfeng
Chemical Reagent (China). Poly (Ethylene Glycol) (Mn =
400 Da) was purchased from Greagent (China).
2.2 Preparation of the microfluidic device and microfibers
The microfluidic device was built
up by coaxially aligning two cylindrical capillaries (1 mm outer
diameter (O.D.) and 0.58 mm inner diameter (I.D.)) inside a square
capillary (1 mm I.D.) according to
previous reports (Cheng et al., 2014; Othman et al., 2015; Yu et al.,
2018). First, the square capillary was fixed onto a glass slide with
epoxy glue. The cylindrical capillary was tapered using a capillary
puller (PC-100, Narishige, Japan) and subsequently shaped using a
microforge (MF-900, Narishige, Japan) to desired orifice (60 or 150 µm
I.D.). Then, the tapered cylindrical capillary was inserted into another
untapered cylindrical capillary in the fixed square capillary on the
glass slide. At last, two syringe needles (2.5 mm O.D. and 0.9 mm I.D.)
with plastic hubs were placed onto the junction between the cylindrical
capillaries and square capillary followed by sealing the gap with epoxy
resin.
The alginate (2%, w/v) solution was pumped using a syringe pump
(SLP01-02A, Longer, China) into the tapered round capillary while the
outer phase (50% PEG and 2% CaCl2) was pumped by
another pump (SLP01-01A, Longer, China) in the identical direction
through the region between the inner cylindrical capillary and outer
square capillary. Straight microfibers were easily fabricated in the
channel via the effective and rapid crosslinking of
Ca2+ and alginate (Yu et al., 2017). The diameter and
composition of alginate-based microfibers were readily modulated by
adjusting the diameter of tapered orifice, flow rate, and the
concentration of the inner or outer phases (Yu et al., 2018). Free BSA,
enzymes, or their conjugates with PAA were added to alginate solution
for fabricating protein/enzyme-loaded microfibers using similar
protocol. Microfibers were collected by wrapping on paper clips for
catalysis assays.
Encapsulation of BSA in
microfibers
2.3.1 Encapsulation efficiency of BSA
BSA-loaded microfibers were prepared using the internal phase
containing alginate (2%, w/v) and BSA (5, 10, 15, 20, 25, 30, 35, 40
mg/mL). The mixture of BSA and alginate was centrifuged (4000 rpm, 1
min) to remove air bubbles prior to the preparation of microfibers.
BSA concentration was determined by the Bradford assay method.
The encapsulation efficiency
(EE) and loading capacity (LC) were calculated according to equations
(1) and (2) (Kahya & Erim, 2019):\(\ EE\%=\frac{m0-m1}{m0}\ \times\ 100\) (1)\(\ LC\%=\frac{m0-m1}{m0+m2-m1}\times 100\) (2)
Where m0 is the feeding mass of BSA in the preparation
of microfibers, m1 is the mass of free BSA,
m2 is the mass of alginate in microfibers.
2.3.2 BSA release studies
BSA-loaded alginate microfibers
were prepared under selected conditions (the flow rate of inner phase:
0.2 mL/h, the flow rate of out phase: 17 mL/h) and collected for 10 min.
Microfibers were immersed in 1 mL deionized water in microcentrifuge
tube and shaken (90 rpm) at room temperature. At each time interval (60
min), 20 µL release media was taken for BSA concentration assays, to
which 20 µL fresh water was added to the centrifuge tube to keep the
volume constant (Kahya & Erim, 2019). BSA content in the samples was
determined by the Bradford protein assay at 595 nm and measurements were
performed with a microplate reader (spectraMAX M5, Molecular Devices,
USA). All experiments were performed in triplicate.
Synthesis of enzyme-polymer conjugates
Carboxyl groups of PAA were activated by EDC/NHS coupling reaction
followed by the addition of GOX for the covalent attachment of enzymes
to PAA (Grabovac et al., 2015; Riccardi et al., 2014; Zore et al.,
2017). Specific steps are as follows: a stock solution of PAA was
prepared by dissolving 0.893 g PAA (35%) in 7 mL deionized water. 0.833
g EDC and 0.25 g NHS were dissolved in 1 mL water and added dropwise
into the PAA solution. The mixture was gently stirred at room
temperature for 18 h after the pH was adjusted to 7.4 by adding
triethylamine. 1 mL GOX solution (10 mg/mL) was added dropwise into the
above mixture and the mixture was stirred for 6 h. GOX-PAA conjugates
solution with GOX concentration of 1 mg/mL was successfully prepared
after dialysis with cellulose membrane (molecular weight cut-off of 200
kDa) against deionized water for the following studies (Ji et al., 2017;
Zore et al., 2017). Other
enzyme-PAA conjugates (HRP-PAA) were synthesized using the same protocol
and mixed with alginate for the fabrication of enzyme-immobilized
microfibers (Riccardi et al., 2014). The covalent conjugates were
confirmed with FTIR spectroscopy (Nicolet 6700 FTIR-ATR analyzer, Thermo
Fisher Scientific, USA). The spectra were recorded in the range of
4000–400 cm-1 with a resolution of 1.0
cm-1 at room temperature.
2.5 Characterization of microfibers
The formation of microfiber was monitored using an optical microscope
with digital camera (LW300LT, Cewei Optoelectronics Technology Co.,
Ltd., China). The diameter of microfibers was measured using the
included software of the microscope. FITC-labeled BSA (FITC-BSA) was
prepared according to previous reports (Kizilay et al., 2014; Zhang et
al., 2010). Fluorescence images of the FITC-BSA encapsulated microfibers
were recorded using an Axio Observer A1 fluorescence microscope (Carl
Zeiss Inc., Germany). The morphologies of fibers were observed using
Scanning Electron Microscopy (GeminiSEM500, Zeiss, Germany). The SEM
images were taken using in-lens detector operating at an accelerating
voltage of 5 kV and a working distance of 5.5 mm.
2.6 Activity assay of enzymes
2.6.1 Activity assay of GOX
GOX catalyzes the conversion of glucose to glucuronic acid
(C6H12O7), producing
hydrogen peroxide (H2O2) simultaneously.
Upon reacting with H2O2, the colorless
substrate TMB is readily converted to the oxidized TMB (OxTMB) which has
a maximum absorption at 652 nm (Lin et al., 2014; Singh et al., 2017).
Hence, the catalytic activity of GOX was evaluated by the measurements
of the consumption of glucose based on these reactions. Specific steps
are as follows: 0.2 mL of glucose solution (5 mM), 0.02 mL of HRP
solution (10 mg/mL), and 2.15 mL phosphate buffer (10 mM, pH 6) were
mixed in a glass cuvette followed by the quick addition of 0.05 mL of
the GOX enzyme solution (0.5 mg/mL) or immobilized enzyme. After 2
min-equilibration, UV absorption (652 nm) was measured. A unit of GOX
activity is defined as the amount of enzyme which oxidizes 1.0 μmol of
β-D-glucose to gluconic acid and H2O2per minute (Wang et al., 2011).
2.6.2 Activity assay of HRP
HRP converts guaiacol to tetraguaiacol (maximum UV absorption at 470 nm)
in the presence of H2O2. The activity of
HRP can be evaluated by measuring the absorbance change at 470 nm
(El-Naggar et al., 2021; Felisardo et al., 2020; Liu et al., 2021).
Specific steps are as follows:
2.85 mL of phosphate buffer solution, 0.05 mL of
H2O2 solution (0.3 %), and 0.1 mL of
guaiacol solution (0.02 mol/L) were added in a glass cuvette (1 cm
width) followed by the quick
addition of 0.05 mL of the enzyme solution or immobilized HRP. The UV
absorption (470 nm) of the mixture was recorded after equilibrating for
2 min. The activity unit of HRP is defined as the amount of the enzyme
which increases the absorption value by 1.0/min under corresponding
assay conditions (El-Naggar et al., 2021).
2.7 Evaluation of the enzyme-immobilized microfibers
2.7.1 pH dependent catalysis
The GOX-PAA immobilized microfibers were collected for 10 min using the
following parameters: the tapered orifice was 60 µm, the flow rate of
inner phase (2% alginate, 0.5 mg/mL GOX-PAA) and outer phase (50% PEG,
2% CaCl2) were 0.2 mL/h and 17 mL/h, respectively. The
activity of free and immobilized GOX-PAA was tested in different buffers
(10 mM, pH 4.0–9.0) at room temperature to assess the optimum pH. The
relative activity of enzyme-immobilized microfibers at varying pHs was
normalized to that of microfibers with the optimum pH.
2.7.2 Temperature dependent catalysis
The enzyme-immobilized microfibers were collected for temperature
dependent assay using the parameters mentioned above. The temperature
dependent catalysis of free and immobilized GOX-PAA were assayed at
40-70 °C to assess the optimum temperature. For the thermal stability
studies, the microfibers or free enzymes were incubated at 40 °C, 50 °C,
60 °C, or 70 °C for 30, 60, or 90 min, followed by immediately cooling
to room temperature for catalytic activity assays. The relative activity
of microfibers was normalized to that of without thermal treated
microfibers.
2.7.3 Reusability of immobilized enzymes
The reusability of enzyme-immobilized microfibers means the capability
of the immobilized GOX to be used for repeated cycles. For this study,
microfibers were immersed in a solution
containing 0.2 mL of glucose
solution (5 mM), 0.02 mL of HRP solution (10 mg/mL), and 2.15 mL buffer
(10 mM, pH 6) in a glass cuvette. After the reaction at room temperature
for 10 min, the microfibers were taken out from the cuvette and TMB
solution was added to the reaction mixture. Then microfibers were washed
with deionized water, and added to the fresh reaction mixture
subsequently. The catalytic activity of microfibers was evaluated by the
analysis of the residual glucose in the reaction solution according to
the protocol mentioned above. The relative activity of microfibers at
each cycle was normalized to that at the first cycle.
2.8 Visual detection of glucose using enzyme-loaded microfibers
For the sensitive detection of glucose, the weight ratio of HRP and GOX
in the microfibers was optimized by changing the composition of the
inner phase in the preparation of HRP and GOX-loaded microfibers. The
total enzyme content was kept constant and the weight ratios of HRP to
GOX (WHRP/WGOX) were
8:1, 4:1, 2:1, 1:1, 1:2, 1:4, or
1:8.
0.1 mL TMB DMSO solution (12 mM) was added dropwise to the mixture
containing 0.5 mL alginate (2%, w/v), 0.2 mL GOX, and 0.2 mL HRP (10
mg/mL) in a 5 mL beaker. The blend was gently stirred for 30 minutes
until it became homogeneous, and used as the inner phase. Enzymes and
TMB-loaded microfibers for glucose detection were prepared and collected
using the method mentioned above. For the detection of glucose, 20 µL
glucose solution (10 mM PBS, pH 6) with various concentrations (0, 0.25,
0.5, 1, 2 mM) was added onto the wrapped microfibers. The color of
microfibers readily changed from light golden to blue with varying
degrees, depending on the concentration of glucose.
Results and discussion3.1 Preparation of microfibers and encapsulation of protein/enzyme
Using the home-made microfluidic device, the straight alginate-based
microfibers was formed continuously due to the feasible gelation upon
the rapid crosslinking of alginate with Ca2+(Fig. S1) . The addition of PEG into CaCl2solution increased the viscosity of the external phase, suppressing
the diffusion of alginate flow and benefiting the stable formation of
microfibers (Yang & Guo, 2019; Zhu et al., 2019a). Moreover, the
diameter of alginate microfibers increased from 21.1 to 90.7 µm with
the decrease of the flow rate of outer phase from 28 to 5 mL/h when
the flow rate of alginate was kept constant at 0.2 mL/h and the inner
diameter of orifice was 60 µm (Fig. S2) . Under these
experimental settings, the higher flow rate of the outer phase
stretched the alginate stream at the constant flow rate of inner
phase, leading to smaller diameter of microfibers. Further increasing
the flow rate of the outer phase ultimately resulted in the break of
microfibers while too low flow rate of the outer phase led to clogging
the microfluidic channel. Thicker microfibers could be fabricated
using the microfluidic device with larger orifice dimension under
proper conditions. For example, microfibers with the diameter of 100
µm could be prepared when the orifice was 150 µm, the flow rate of
outer phase and inner phase were 18 and 0.5 mL/h, respectively. It was
obviously that fluids mainly form laminar flow under the above
conditions.