Page 3 of 14
1
Analytical Chemistry
with brine (200 mL), dried over anhydrous Na
concentrated and purified by flash column (C18 reversed phase,
MeCN/H O as eluent) to give product 3 (3.0 g, yield 47%) as a
2
SO
4
,
To determine the influence of the enzymatic reaction time on
the results, enzymatic reaction times of 5, 10, 20 and 30 min
were tested, and other procedures were as described above.
2
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9
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6
2
yellow solid.
For the specificity of the immunoassay, seven control
experiments were performed. Three common interference
components (CRP, IL-6 and human IgG) and a mixture of PCT
and blank samples were selected to investigate the selectivity
and specificity of the ACF immunosensor. The concentrations
Then, the product 3 (3.0 g, 4.3 mmoL) was dissolved in DCM
o
(90 mL) and cooled to 0 C, and TFA (trifluoroacetic acid, 9
mL) was added dropwise, and the resulting mixture was stirred
at RT for 0.5 h. The mixture was concentrated at RT under
reduced pressure and the residue was triturated with
-1
of CRP, IL-6 and human IgG were 100 ng mL in PBS buffer,
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
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0
-1
CH
3
CN/DCM (v/v = 1/3, 40 mL) and filtered to afford
and the concentration of PCT was 7.78 ng mL . Employing the
compound 4 (MX-66, 500 mg, yield 20%) as a yellow solid.
1H NMR (500 MHz, DMSO-d
J=8.5Hz, 1H), 8.04 (d, J=8.0Hz, 1H), 7.97 (s, 2H), 7.76 (t,
J=7.0Hz, 1H), 7.70-7.50 (m, 1H), 7.52 (s, 3H), 7.44 (t, J=7.5Hz,
1H), 7.24-7.15 (m, 5H), 5.52-5.48 (m, 1H), 4.93-4.70 (m, 2H),
4.74 (d, J=4.8Hz, 1H), 3.53-3.40 (m, 4H).
13C NMR (126 MHz, DMSO-d
163.64, 156.39, 155.30, 139.74, 136.32, 134.63, 131.41,
130.82, 129.49, 129.17, 129.11, 128.68, 126.95, 125.59,
same experimental procedures described above, the seven
samples were assayed, and each sample was repeated three
times.
6
) δ 13.66 (s, 1H), 9.01 (d,
Spiking recovery of PCT in human serum sample. To
remove the adipose tissue, the healthy human serum was
centrifuged at 10000 rpm for 20 min three times. Then, 50 μL
of PCT at various concentrations (0.432, 1.30, 3.89, 11.7, 35.0
6
) δ 174.39, 171.46, 164.77,
-1
and 70.0 ng mL ) was spiked in 350 μL of the above
centrifuged serum. The spiked samples were assayed with the
procedures described above. The final concentrations of PCT
123.96, 118.93, 71.25, 59.37, 57.94, 42.03, 40.48, 40.31, 40.15,
-1
+
were 0.273, 0.547, 1.09, 2.19, 4.38 and 8.75 ng mL .
3
9.98, 39.81, 39.65, 39.48, 26.31. HRMS (ESI ) m/z calcd for
+
C
31
H
24
N
2
O
7
S [M] 568.13042, found 569.13802.
MX-66: (6R,7R)-8-oxo-3-(((4-oxo-2-phenyl-4H-chromen-
-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo
Real patient samples detection. The PCT levels in 14 real
patient serum samples (obtained from Beijing Friendship
Hospital with patient consent) were detected with the ACF
immunosensor following the procedures described above, and
each sample was assayed three times. For verification, a
commercial ELISA kit was also utilized to detect the PCT levels
of the same 14 samples with the recommended method of the
kit. Briefly, 50 μL of standard sample or real sample was added
to the 96-well plates along with 100 μL of HRP-labeled PCT
detection antibody. The 96-well plates were incubated at 37°C
for 1 h and washed five times with washing buffer, substrates A
and B were added to the plates, and the plates were incubated at
3
[4.2.0] oct-2-ene-2-carboxylic acid.
We commissioned 9dingchem Co., Ltd to synthesize the
MX-66.
Characterization of the ACF immunosensor. The
detection of PCT was carried out as follows: The Ab
conjugated immuno-magnetic balls (IMBs-Ab ) complex and
the Ab and AmpC-conjugated polystyrene microspheres (PS-
Ab /(AmpC) ) complex (nAmpC/nAb2=100:1) were first diluted to
.25 mg mL and 1.0 mg mL with PBS buffer, respectively.
1
-
1
2
2
n
-
1
-1
0
3
7°C for 15 min. Finally, 50 μL of the stopping solution was
-1
Then, 50 μL of 0.25 mg mL IMBs-Ab
into 450 μL of PCT solution with various concentrations
1
complex were added
added to the plates, and the UV absorbance of the samples was
detected at 450 nm.
-1
(
0.0960, 0.288, 0.864, 2.59, 7.78, 23.3 and 70.0 ng mL ,
respectively) and reacted on a roller for 20 min at 37°C. Then,
the IMBs-Ab -PCT complex was separated by a magnetic
Reagents and instruments, and other experiments procedures
were described in supporting information.
1
separator and washed twice with 500 μL PBST buffer and
resuspended in 500 μL PBS buffer. Fifty microliters of 1.0 mg
■
RESULTS AND DISCCUSSION
-1
Design and synthesis of the substrate for the AmpC (MX-
2 n
mL PS-Ab /(AmpC) complex was subsequently introduced
6
6). To develop an enzymatic reaction system, the substrate for
and reacted on a roller for 20 min at 37°C. The unreacted
complex was removed by a magnetic separator, and the double
sandwich complex was washed with 500 μL PBST buffer three
times and transferred to a new tube in which 400 μL of MX-66
solution (1.0*10 mol L ) was added, and the mixture solution
was incubated for 20 min on a roller at 37°C. Then, the double
sandwich complex was removed by a magnetic separator, and
24 2 7
the AmpC enzyme was designed as MX-66 (C31H N O S),
which contained the β-lactam ring and cephalosporin ring as the
enzymatic recognition section and the structural motif of a
natural green dye, 3-HF, as the reporter molecule. The synthetic
route of MX-66 is shown in Fig. 1.
-4
-1
The structure of the product was confirmed by high-
resolution mass spectrometry (HRMS, Fig. S2), Fourier
transformed infrared spectroscopy (FTIR, Fig. S1 and Table S1)
and nuclear magnetic resonance (NMR, Fig. S3-S5)
approaches. In the HRMS spectra of the product, there are two
main peaks at 569.13802 Da (m/z) and 591.12004 Da (m/z),
-1
25 μL of β-CD-ZnS QDs (0.711 mmol L ) was introduced into
the solution. The fluorescence intensities at 530 nm were
collected at an excitation wavelength of 350 nm. The final
concentrations of MX-66 and β-CD-ZnS QDs were 75.5 μmol
-1
-1
L and 44.4 μmol L , respectively.
+
+
which represent the M+H and M+Na , respectively. From the
exact mass and isotopic profiles in the HRMS spectra, the
With similar procedures, the concentration of PCT was fixed
-
1
to 1.40 ng mL , and the influence of the PS-Ab
complex with various molar ratios of AmpC to Ab
surface of PS microspheres (nAmpC/nAb2=30:1, 70:1, 100:1,
30:1, 160:1, respectively) on the fluorescence intensities was
investigated.
2
/(AmpC)
n
24 2 7
molecular formula of the product was deduced as C31H N O S,
2
on the
which was the same as that of the designed target MX-66 (the
theoretical exact mass of the target MX-66 is 568.13042 Da.).
The characteristic FTIR absorption bands of the main functional
groups are described in Table S1, and these findings suggested
1
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