M. Ray et al.
Analytical Biochemistry 626 (2021) 114204
(Sephadex G-25, Amersham) to remove the glycerol and camphor
(wherever necessary) before experiments.
3. Results and discussion
3.1. Characterization of p-CPMI
2.5. Preparation and characterization of the bio-nano conjugates
P-CPMI synthesized by the earlier reported method [23]was char-
acterized by FTIR and NMR spectroscopy. 1HNMR spectrum of p-CPMI
in deuterated DMSO shows peaks at 13.06 δ(s, 1H, COOH), 8.05 δ (d,
2H, ortho COOH), 7.5 δ (d, 2H, ortho to imide nitrogen), 7.2 δ(s, 2H,
olefinic), which agree with reported values [23]. The FTIR spectrum of
p-CPMI (Fig. S3a in Supplementary information) showed two peaks at
1710 cmꢀ 1 and 1775 cmꢀ 1 corresponding to the carbonyl stretching
SWCNT conjugated maleimide (SWCNT-CPMI) was ultra-sonicated
in DMF. 200
μl of SWCNT-CPMI (10 mg/mL) dispersed DMF was
mixed with 50 mM phosphate buffer at pH 7.4 containing 1 mM
camphor to a final volume of 2 mL 500 l of concentrated protein so-
μ
lution was added to the 2 mL of the above solution and incubated for 30
min at 4 ◦C to form CYP101 conjugated to SWCNT-CPMI (SWCNT-CPMI:
CYP101). The unreacted protein was removed by size-exclusion chro-
matography using PD-10 column. The protein conjugated to SWCNT-
CPMI:CYP101 collected by size-exclusion chromatography was used
for UV–visible absorption, Circular Dichroism (CD) and AFM
experiments.
–
–
–
frequency of O C–N–C O bond of maleimide (as maleimide has a five
–
–
membered cyclic amide ring and C C double bond). The FTIR spectrum
–
of p-CPMI also shows broad peak at ~3477-2550 cmꢀ 1 due to –OH
stretching of carboxylic acid. The peak at 1600 cmꢀ 1 is assigned to
carbonyl group of carboxylic acid and that at 1215 cmꢀ 1 is due to C–O
stretching of carboxylic acid present in p-CPMI. There is also a peak at
~1515 cmꢀ 1, which arises due to the aromatic ring in p-CPMI.
UV–Vis absorption spectra of the protein solution were measured
using Perkin–Elmer λ-750 instrument. Concentration of CYP101 in the
protein solution was determined from the absorbance at 391 nm of the
enzyme in presence of the substrate (ε391 = 102 mMꢀ 1cmꢀ 1). The con-
centration of the enzyme conjugated to the SWCNT-CPMI was deter-
mined by deconvolution of the UV–visible absorption spectrum of an
aliquat of the solution of SWCNT-CPMI:CYP101 with that of free
SWCNT-CPMI (Fig. S2 in Supplementary information).
3.2. Covalent linkage of maleimide to SWCNT
Carbonyl carbon of carboxylic group of p-CPMI can be covalently
linked to SWCNT by treating with polyphosphoric acid and P2O5 at
130 ◦C for 3 days in inert atmosphere. Scheme 1 shows the conjugation
of SWCNT with p-CPMI, which follows a Friedel–Crafts’s type acylation
reaction [25–29] mechanism as shown earlier. In the present case pol-
yphosphoric acid was used as the Lewis acid catalyst. In order to
determine conjugation of p-CPMI with the nanotube, the FTIR spectrum
of p-CPMI conjugated SWCNT was obtained (Fig. S3b in Supplementary
information). Comparison of the FTIR spectrum of pure CPMI (Fig. S3a
in Supplementary information) with that of p-CPMI linked to the SWCNT
(Fig. S3b in Supplementary information) showed presence of the broad
peak ~1710 cmꢀ 1 (shown by vertical dotted line in Fig. S3 in Supple-
mentary information) corresponding to the carbonyl stretching fre-
All CD spectral measurements were performed using a JASCO-810
polarimeter. The tertiary structure of CYP101 was measured by
recording the CD spectra of 20
μM enzyme in potassium phosphate
buffer (pH 7.4) in 250–700 nm range using quartz cuvette of 1 cm path
length. Concentration of the enzyme in SWCNT-CPMI:CYP101) was 0.6
μ
M. The experiments were also carried out at room temperature unless
stated otherwise.
Atomic force microscopy (AFM) was done using a PicoScan (Mo-
lecular Imaging USA) setup in non-contact AC mode with a liquid cell. A
silicon cantilever tip with a resonance frequency of 80 kHz and spring
constant 5 N/m was used for imaging the sample and the images were
analyzed using standard image analysis software (SPIP, from Image
Metrology, Denmark). The particle heights were measured at random
intervals from several image scans of the sample.
–
–
–
quency of O C–N–C O bond of maleimide in the SWCNT-CPMI
–
conjugate. However, the intensity of the peak is decreased on conjuga-
tion on SWCNT. The carbonyl group of the carboxylic acid of p-CPMI
shows a peak at ~1600 cmꢀ 1. The peak (Fig. S3a in Supplementary
information) at 1215 cmꢀ 1, present in p-CPMI due to C–O stretching
frequency of carboxylic acid is absent (Fig. 3b in Supplementary infor-
mation) in modified SWCNT, supporting that the carboxylic acid group
of CPMI participates in the linkage with SWCNT as shown in Scheme 1
[26]. The peak at 1515 cmꢀ 1 due to aromatic ring in p-CPMI is shifted to
1509 cmꢀ 1 in functionalized SWCNT. The peaks around 3200 to 3400
cmꢀ 1 region in SWCNT-CPMI are due to the –OH stretching of carboxylic
acid of SWCNT (Fig. S3c in Supplementary information). Thus, the FTIR
data signifies that the p-CPMI is covalently conjugated to SWCNT. It is
also seen that dispersed solution of p-CPMI conjugated SWCNT (Fig. S4B
in Supplementary information) in DMF is stable for long time (5–10
months) while dispersed unmodified SWCNT (Fig. S4A in Supplemen-
tary information) in DMF settles down immediately supporting that the
conjugation of p-CPMI with SWCNT increases the polarity of the nano-
tubes making them more stable in the solution.
Cyclic voltammetric experiments were carried out at room temper-
ature using Autolab potentiostat-30 instrument in a three-electrode as-
sembly with Ag/AgCl (3 M KCl) as the reference electrode and a
platinum wire as the counter electrode to GC as working electrode. Ni-
trogen gas was purged through 10 mL solution for at least 10 min to
remove any dissolved oxygen before every experiment. Nitrogen atmo-
sphere was maintained over the solutions during experiments. Prior to
every experiment, suitable pre-treatment of the working electrode was
carried out. Pre-treatment of GC electrode involves polishing firmly on
micro-cloth using fine 0.05 μm alumina powders. It was then ultra-
sonicated for 1 min in Milli Q water, rinsed thoroughly with water, and
used immediately. The GC electrode was dipped in the SWCNT-CPMI
solution in DMF for overnight. The modified electrode was washed by
dipping in Milli Q water and subsequently dipped in 1% TritonX-100
solution for 10 min before washing again by Milli Q water. The
SWCNT-CPMI modified GC electrode then dipped in CYP101 solution
Raman spectroscopy has been used to characterize the functionalized
SWCNTs. The inset of (Fig. S5 in Supplementary information shows
Raman spectra of SWCNT and SWCNT-CPMI. Each spectrum showed
two peaks known as D band and G band. The D band found near 1300
cmꢀ 1 is used to evaluate the defect density present in the tubular wall
structure and the G-band in the 1550–1600 cmꢀ 1 region of spectrum is
due to the [30,31] tangential C–C stretching of SWCNT carbon atoms.
The ratio of D/G band intensity (ID/IG) depends on the sp2 hybridized
carbon content. Bare SWCNT shows strong G band at 1573 cmꢀ 1 is
Raman allowed phonon high frequency mode and the D band at 1321
cmꢀ 1 originates from defects in the sites on nanotube surface. After
functionalization, the peak positions in Raman spectra and relative in-
tensities of G and D band change slightly. The ratios of the intensities of
(20 μM) overnight for formation of the SWCNT-CPMI:CYP101 bio con-
jugate on the electrode. The bio-nano conjugate SWCNT-CPMI:CYP101
prepared by reaction of the protein with SWCNT-CPMI in solution also
showed analogous electrochemical response when drop casted on the
GC electrode from a solution containing 1% TritonX-100. The working
buffer was 40 mM potassium phosphate, 50 mM KCl, pH 7.4. The
electrode potential values were reported with respect to the Ag/AgCl
electrode (3 M KCl).
3