Bioimmobilization Matrices with Ultrahigh Efficiency Based on

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Bio-Immobilization Matrices with Ultra-High Efficiency Based on Combined Polymerizations of Chemical Oxidation and Metal Organic Coordination for Biosensing Xin Qi, Minrui Li, Yingchun Fu, Chunyang Lei, Yanbin Li, Qingji Xie, and Shouzhuo Yao J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b01278 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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The Journal of Physical Chemistry

Bio-Immobilization Matrices with Ultra-High Efficiency Based on Combined Polymerizations of Chemical Oxidation and Metal Organic Coordination for Biosensing

Xin Qi,a,b Minrui Li,a Yingchun Fu,a,b,* Chunyang Lei,a Yanbin Li,a,d Qingji Xie,b and Shouzhuo Yaob,c

a

College of Biosystems Engineering and Food Science, Zhejiang University,

Hangzhou 310058, China. b

Key Laboratory of Chemical Biology and Traditional Chinese Medicine

Research (Ministry of Education of China), Hunan Normal University, Changsha 410081, China. c

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of

Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China d

Department of Biological and Agricultural Engineering, University of Arkansas,

Fayetteville, AR 72701, USA

1

ACS Paragon Plus Environment * Corresponding author. Tel./Fax: +86 571 88982534. E-mail address: [email protected].


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ABSTRACT

Facile regulation and enhancing of the performance of bio-immobilization materials is a key factor for their applications for biosensing, biocatalysis, bioreactor, and so on. Here, we propose a method of combined polymerizations of chemical oxidation and metal organic coordination to develop enhanced bio-immobilization matrices for high performance biosensing. Being different from conventional methods that based on sole polymerization, the new method elaborated chemical oxidation to one-pot obtain oligomers as ligands for metal-organic coordination polymerization. Two kinds of thiol that could be chemically oxidized by H2O2 and be coordinated with NaAuCl4 were adopted as monomers. Glucose oxidase was adopted as the representative biomolecule. Chemical oxidation was proved to be efficient to lengthen monomers to produce oligomers (ligands) with different length by adjusting the concentrations of monomers and oxidant, as well as reaction time. This dynamic pre-lengthening process not only endows the co-existing biomolecules with active and protective oligomers shell to significantly enhance the immobilization efficiency, but also regulates the structure of metal-organic coordination polymer. As crucial factors of immobilization, the entrapment ratio of enzyme and mass transfer efficiency all achieved obvious increases comparing with those based on sole chemical oxidation polymerization or metal-organic coordination polymerization, the entrapment ratio even reached an extreme value of 100%. Therefore, the biosensing performance was greatly promoted with sensitivities being among the best of those reported analogues. 2

ACS Paragon Plus Environment

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The biosensors also exhibited satisfactory selectivity, stability and feasibility for blood serum samples. This method may provide a universal strategy for regulating and enhancing performance of ligand-constructed polymers and their composites for entrapment-based applications.

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INTRODUCTION Metal organic coordination polymers (MOCPs) are an emerging class of inorganic-organic hybrid materials, such as metal organic frameworks (MOFs) and infinite coordination polymers (ICPs),1-2 and have been broadly explored for numerous

potentials,

including

energy

storage,3

chemical

separations,4

chemical-/bio-sensing,5-7 catalysis,8-9 and drug delivery.10-11 In most applications, the design and regulation of structure and performance is the key but difficult issue, which leads to plenty of efforts on the synthesis of organic ligands with desired structure (e.g. length) and functional groups. However, this common strategy based on preformed ligands lacks of flexibility to regulate the structure and performance of a designed product during the polymerization. Additionally, once the ligands are preformed, so are the interactions between the ligands and co-existing species, making it hard to further introduce new or enhance interactions, which is an obvious limitation especially for the increasingly important applications of MOCPs as encapsulation matrice.6, 12-17 Therefore, introducing one-pot dynamic synthesis of the monomer/ligand before polymerization may create a new strategy for regulating and enhancing the performance of MOCPs. The immobilization of biomolecules is one of the key issues in academic researches and industrial applications, such as biosensing, bio-reactor, and drug delivery.6, 13, 15, 18-22

To evaluate the immobilization efficiency, the load/activity of the immobilized

biomolecules and the mass-transfer efficiency of the relevant species are crucial.6, 19 Generally, there are 2-D and 3-D strategies.19 2-D protocols offer favorable mass transfer efficiency but often suffer from limited load and activity, since 2-D surface area is limited and the un-protected biomolecules are high susceptible to environment.20, 23 In contrast, the 3-D protocols can provide more and protected sites for biomolecules,24-25 however, the mass-transfer efficiencies are suppressed. More deeply, general strategies are based on the directly involving of biomolecules on the interface of matrix, leading to limited conjugation/anchoring sites between the

4


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surfaces of biomolecule and matrix, as well as unavailable loss of activity due to the conformation change under mechanical and/or chemical effects. Therefore, novel strategy that can enhance the interactions between matrix and targets, as well as provide effective protection on the targets and sufficient mass transfer, should be promising to satisfy the demands on the three factors of high efficient immobilizations. 2

1

Monomer

NaAuCl4 Path I

Oligomer

H2O2 3

Enzyme

Path II

4

MOCPs

NaAuCl4

Scheme 1. Illustration of the preparation of the MOCPs and MOCPsco. Herein, we report on a novel method to regulate the performance of MOCPs through one-pot chemical oxidation of monomers to lengthen ligands to develop immobilization matrices with ultra-high efficiency. 1,4-Benzenedithiol (BDT) and 2,5-dimercapto-1,3,4-thiadiazole (DMcT) were utilized as monomers and ligands. H2O2 as a metal-free oxidant was used to chemically oxidize the -SH of BDT and DMcT to form BDT oligomer and DMcT oligomer via the S-S linking (Schemes S1).26-27 NaAuCl4 as a metallic connector could coordinate with the chemical oxidation-yielded oligomers (the ligands) to form MOCPs (MOCPsco) (Scheme 1, path II, from 3 to 4). Glucose oxidase (GOx) was used as a model protein to evaluate the immobilization efficiency and biosensing performance. As illustrated in Scheme 1 for the mechanism of the immobilization, the chemical oxidation produced oligomers that were unstable and in turn in-situ adsorbed onto the surface of enzyme to form a shell, which provided more coordination (anchor) sites for further immobilization and protection on enzyme from activity loss during the following metal organic coordination polymerization. The un-adsorbed oligomers could also further participate 5



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in coordination polymerization to regulate the structure (porosity) of MOCPs. Compared with MOCPs without the chemical oxidation (Scheme 1, path I), MOCPsco presented higher immobilization efficiency to enzyme and higher mass transfer efficiency of H2O2, as well as superior biosensing performance. The entrapment ratio even reached 100% in the BDT case. EXPERIMENTAL SECTION Materials and Apparatus All electrochemical experiments were conducted on a CHI660C electrochemical work-station (CH Instrument Co.) with a conventional three-electrode electrolytic cell. The Au electrode with 2.0 mm diameter (area =0.03 cm2, Tianjin Incole Union Technology Co., Ltd) served as the working electrode, a KCl-saturated calomel electrode (SCE) as the reference electrode, and a carbon rod as the counter electrode. All potentials reported here are cited versus SCE. UV-vis spectrophotometry was conducted on a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan) and a Synergy H1 Hybrid Multi-Mode Microplate reader (BioTek, Vinooski, VT, USA). Scanning electron microscopy (SEM) pictures were collected on a JEM-6700F field emission scanning electron microscope (JEOL, Japan). GOx (EC 1.1.3.4; type II from Aspergillusniger, 150 kU g-1) was purchased from Sigma. DMcT and BDT (97%) were purchased from Alfa Aesar. A pH 7.0 phosphate buffer solution (PBS, 0.1 M KH2PO4/K2HPO4 + 0.1 M K2SO4) was used. Blood serum samples were gifted kindly by the Campus Hospital of Zhejiang University. All

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other chemicals were of analytical grade or better quality, and used as received. Milli-Q (Millipore, ≥18 MΩ cm) ultra-pure water was used throughout. Preparation of different polymeric biocomposites. Several biocomposites were prepared through different polymerization ways as follows. In the MOCPsco cases, to an ultrasonically dispersed aqueous of 1.0 mg mL-1 BDT or DMcT in 1 mL of PBS, 4.5 mg of GOx was added under stirring, and then 0.2 mM H2O2 (BDT case) or 0.75 mM H2O2 (DMcT case) (both ﬁnal concentrations) was slowly added. The mixture was stirred to allow a 10-min reaction to produce oligomers and form oligomers-GOx composites. Afterwards, 5 mM NaAuCl4 was added to form MOCPsco. In the conventional metal-organic coordination enzyme cases, procedures were the same except the absence of chemical oxidation. For the chemical oxidation biocomposites case, in an ultrasonically dispersed solution of 2.5 mg mL-1 BDT (or 3 mg mL-1 DMcT) in 1 mL PBS, 4.5 mg GOx was added under stirring, and then 5 mM H2O2 (final concentration, 15 mM for DMcT) was slowly added. The mixture was stirred to allow 30 min to obtain chemical oxidation-based BDT

polymer

(PBDT)-GOx

or chemical oxidation-based DMcT polymer

(PDMcT)-GOx biocomposites. Preparation of the electrodes modified with biocomposites. The Au electrode was carefully cleaned and was immediately used for modification.28 The biocomposites suspensions prepared above were centrifuged, the precipitates were successively water-rinsed for three times, re-dispersed in 200 µL

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water, and dip-dried on a clean Au electrode, followed by water evaporation in air. When not in use, the modified electrodes were stored in pH 7.0 PBS at 4 oC. Evaluation of the entrapment ratio of enzymes based on Bradford method. The protein assay reagent was prepared according to previous report.29 Good linear relationship (r2 > 0.995) was obtained using the protein assay reagent to quantify GOx standard solutions. After the polymerization of different biocomposites, the centrifugation-isolated supernatant (20 µL) was diluted with 70 µL PBS. Twenty µL diluted supernatant was then mixed with 1 mL protein assay reagent. After 5-min reaction, 200 µL final solution was used to collect UV-vis spectra and the absorbance at 595 nm was adopted for comparison. GOx solution of 4.5 mg mL-1 was used as the control based on the same processes. Amperometric Biosensing Measurements. Measurements of the biocomposites-modified electrodes were carried out under solution-stirred conditions in pH 7.0 PBS, and the response current was marked with the change value between the steady-state current after addition of a substrate and the initial background current without the substrate. RESULTS AND DISCUSSION Fabrication and Characterization of Different Biocomposites Digital imaging was used to visually investigate the different ways of polymerization, as shown in Figures 1 and S1 (set BDT polymerization as an example). Assisted by ultra-sonication, BDT (sample 1) and its mixture with GOx (sample 2) formed good suspension in PBS. After the addition of H2O2 into the sample 2, the mixture gradually deepened its yellow colour and became turbid, finally 8


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Figure 1. Digital picture of several BDT-containing PBS suspensions (0.55 mL) containing BDT (1), BDT + GOx (2), PBDT-GOx (3), BDT-MOCPs (4), and BDT-MOCPsco (5). Concentrations: BDT, 1.5 mg mL-1 for suspension 3 and 1 mg mL-1 for others; GOx, 4.5 mg mL-1; H2O2 5 mM for suspension 3 and 0.2 mM for suspension 5; NaAuCl4, 5 mM. The arrows indicate the precipitates in samples 3. formed a few white-coloured precipitates in the bottom. The colour and turbidity changes reported the gradual elongation of monomers to oligomers/polymers through chemical oxidation polymerization. It is well known that the longer the length of oligomer chain, the poorer the solubility, which means oligomers have strong driving force to adsorb onto co-existing species (such as enzyme) to stabilize themselves. This gradually endowed enzyme with a shell of oligomers, which should in turn protect enzyme and introduce much more active sites (such as residual SH groups) for further coordination polymerization than the enzymes themselves do. For the direct coordination polymerization, the direct addition of NaAuCl4 to the sample 2 yielded lots of precipitates, which were the products of the coordination of Au with S (sample 4), as reported in our previous report.6 For the MOCPsco case, the addition of H2O2 to sample 2 caused the colour little deeper (limited chemical oxidation), and the following addition of NaAuCl4 rapidly produced lots of brown-coloured loose precipitates (sample 5). Additionally, the supernatants of samples 4 and 5 were obviously clearer than that of samples 2 and 3, which implied most of enzymes with yellow colour had been entrapped in the precipitates. Similar results were obtained in the DMcT case. Note that monomers and oligomers were used as the ligands for sole

9


.6

.6 3

.3

0.0 240

.4

y=0.94 - 0.48x r2=0.9555

.2

270

300

330

Wavelength / nm

B

1.0 .8

t (min) 0

.9 .6 .3

30

0.0 240 270

.6

300 330

Wavelength / nm

y=1.13-0.64lgx 2 r =0.9758

.4

0.0

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1.2

1.2

Absorbance

.8

H2O2 (mM) 0

.9

Absorbance

A

1.0

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Absorbance


.2 0.0

.5

1.0

1.5

2.0

1

2.5

10

100

t / min

cH O / mM 2 2

Figure 2. (A) UV-vis spectra for the absorbance of BDT at 284 nm with different concentrations of H2O2 from 0 mM to 3 mM and calibration curves (insert). The initial concentration of BDT was 0.3 mg mL-1, oxidation time for 10 min. (B) the absorbance of BDT at 284 nm with different oxidation times from 0 min to 30 min and calibration curves (insert). The initial concentration of BDT was 0.3 mg mL-1 and H2O2 was 1 mM. metal organic coordination and the combined polymerization, respectively. Above results demonstrated that the chemical oxidation was efficient to produce oligomers/polymers as ligands for the coordination polymerization to yield porous products. To evaluate how H2O2 controls the chemical oxidation, we investigated the influence of the concentrations of H2O2 and oxidation time using UV-vis spectroscopy. The absorbance peaks of BDT and DMcT monomers at 284 nm

30

and 320 nm,31

respectively, were selected to monitor the transformation of the monomers to the oligomers. As shown in Figure 2(A) for BDT, the absorbance at 284 nm showed a gradual decrease along with the increase of the concentration of H2O2, a linearity was obtained at concentrations from 0 mM to 1.5 mM (r2 = 0.956). This indicated the gradual transformation of BDT monomers to oligomers that was controllable by the concentration of H2O2. Similarly, along with the increase of the oxidation time at a H2O2 concentration of 1 mM, BDT was gradually transferred into oligomer, as shown in Figure 2(B). A linearity (r2 = 0.976) was also obtained at time period from 0 min to 30 min, which means the length of the oligomers could be regulated by the oxidation time too. We finally chose a H2O2 concentration of 0.2 mM and an oxidation time of

10


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1

2

3

1 µm

1 µm

1 µm

4

5

6

1 µm

1 µm

1 µm

Figure 3. SEM images of PBDT-GOx (1), BDT-MOCPs (2), BDT-MOCPsco (3), PDMcT-GOx (4), DMcT-MOCPs (5), and DMcT-MOCPsco (6). 10 min for the most efficient immobilization, the absorption intensities of which were close to that of pure monomer suspensions. This demonstrates that the yielded oligomers should be mainly short ones, which is beneficial to the porosity and performance of MOCPsco, as detailed below. For the DMcT case (as shown in Figure S2), we obtained similar results. Although oxygen is also an oxidant involved in the chemical oxidation, the rate is too slow and the time for reaction (10 min) is too short to interfere the H2O2-induced chemical oxidation, as proved in our previous study.27 Therefore, through controlling the oxidant concentration and oxidation time, the chemical oxidation could be simple and facile way to regulate the length of oligomers (ligands), and in turn to prepare MOCPsco with desired performance. SEM was adopted to characterize the chemical oxidation-based polymers, conventional MOCPs and MOCPsco in both BDT and DMcT cases, as shown in Figure 3. For the chemical oxidation-based polymers, SEM (images 1 and 4) show lumps and pieces of polymers with quite compact structure, which agrees with the observations in Figures 1 and S1. The conventional MOCPs presented porous 3-D microstructures of particles (BDT, image 2) and irregular fibres (DMcT, image 5). For the BDT-MOCPsco (image 3), plenty of nanoparticles with size (ca. 40 nm) being similar as that in the BDT-MOCPs were found. Interestingly for the DMcT-MOCPsco (image 6), nanorods instead of fibres were observed. Here, both MOCPsco clearly

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showed combined characteristics of the structure of the chemical oxidation-based polymers and MOCPs, which proved the combination of two polymerization ways. The entrapment efficiency of GOx in different biocomposites The entrapment efficiencies were evaluated by quantifying the residual GOx in the centrifugation-isolated supernatant of the MOCPs-contained suspension using UV-vis spectrophotometry. Namely, the subtraction of the residual GOx in the supernatant from the initial one should give the entrapped one. Bradford method was adopted for comparison of the concentration of GOx in the BDT case. In the DMcT case, the absorbance of GOx at 280 nm was directly used for quantification because there is no interference existed around this wavelength. As shown in Figure 4 for the BDT case, the increase in the concentration of H2O2 from 0 mM to 0.2 mM or the oxidation time from 0 to 10 min increased the entrapment efficiency. Imaginably, the gradual elongation of the ligands (oligomers) by the chemical oxidation should introduce more immobilization sites and increase the porosity of the MOCPsco to promote the entrapment. However, the further increase of the H2O2 concentration or the oxidation time led to a sharp decrease in the entrapment efficiency. Here, long oligomers could tangle with each other and finally form dense chemical polymers, as shown in Figure 3 (images 1 and 3) which should significantly reduce the entrapment efficiency. Similar results were obtained in the DMcT case, as shown in Figure S3. Therefore, through controlling the concentration of the oxidant and the oxidation time, one could readily regulate the entrapment efficiency using the pre-lengthening method. For comparison, the entrapment efficiencies of GOx in chemical polymer and oxidation-free MOCPs were measured using the same methods for both BDT and DMcT cases. The PBS solution containing GOx and BDT with identical concentrations to those for polymerization was used as the control. As shown in Figure 5(A), there was no obvious absorbance at 595 nm in the supernatant of BDT-MOCPsco, which indicated that nearly all the added GOx were captured in the BDT-MOCPsco. The absorbance of GOx in the supernatants of BDT-MOCPs and 12


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.04

.6

.02 0

1

2

cH O / mM

0.00

2

3

.04

6 1 2 4

2

5

0.2 mM H2O2 400

450

500

550

.8 .03 .02 0 10 20 30 40

7

600

650

6

4

t / min

10 min

0.00

700

3 400

Wavelength / nm

B

5 1 2

.01

3

-.02 350

1.0

.05

Entrapment ratio

.8

.06

A 7

Absorbance

1.0 .08

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Entrapment ratio

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500

600

700

Wavelength / nm

Figure 4. UV-vis spectra for the Bradford protein assay reagent mixed with the centrifugation-isolated supernatant of the suspension of (A) BDT-MOCPs (1) and BDT-MOCPsco with different concentration H2O2. 0.1 mM (2), 0.2 mM (3), 0.5 mM (4), 1 mM (5), 2 mM (6), and 3 mM (7), as well as (B) BDT-MOCPs (1) and BDT-MOCPsco with different oxidation times. 5 min (2), 10 min (3), 15 min (4), 20 min (5), 30 min (6), and 40 min (7). The entrapment ratio calibration curves are given as the insert. All the centrifugation-isolated supernatant of the suspension were 4.5-fold diluted. chemical oxidation polymer (PBDT) were increased to be 0.025 ± 0.005 (n = 3, the same as below) and 0.074 ± 0.008, being 15% and 46% of that in the GOx and BDT mixture case (0.161 ± 0.021), respectively. We calculated the entrapment ratio of GOx in BDT-MOCPsco, BDT-MOCPs, and PBDT were 100%, 85% and 54%, respectively. The enzymatic activity of GOx treated by the chemical oxdiation was also investigated. GOx solutions were incubated in the presence and absence of H2O2, then the enzyme activity was examined using a classic UV-vis spectroscopy method by adding GOx into a reaction solution containing glucose, horseradish peroxidase and o-dianisidine dihydrochloride. Glucose is catalyzed by GOx and yields gluconic acid and H2O2. Then H2O2 was catalyzed by horseradish peroxidase, which turns o-dianisidine dihydrochloride into brown product that can be read directly at 436 nm.26 The enzymatic activity thus could be reflected by the ratio of the adsorbace change considering the identical concentration. As shown in Figure S4, the GOx solutions treated by 0.75 mM H2O2 for 10 min presented almost the same absorbance

13



.5

.15

A

B

1

1

.4

.10

Absorbance

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2

.05

3 0.00 4

.3 2 .2 3

.1

4

-.05 400

450

500

550

600

650

0.0 240

700

260

280

300

320

Wavelength / nm

Wavelength / nm

Figure 5. (A) UV-vis spectra for the Bradford protein assay reagent mixed with the GOx + BDT solution (1) and the centrifugation-isolated supernatant of the suspension of PBDT-GOx (2), BDT-MOCPs (3), and BDT-MOCPsco (4). All the centrifugation-isolated supernatant of the suspension were 4.5-fold diluted before the measurements. Concentrations: BDT, 2.5 mg mL-1 for suspension 2 and 1 mg mL-1 for others; H2O2, 5 mM for suspension 2 and 0.2 mM for suspension 4; NaAuCl4, 5 mM. (B) UV-vis spectra for 4.5 mg mL-1 GOx solution (1) and the centrifugation-isolated supernatant of the suspension of PDMcT-GOx (2), DMcT-MOCPs (3), and DMcT-MOCPsco (4). The initial concentrations of GOx were all 4.5 mg mL-1. Concentrations: DMcT, 3 mg mL-1 for suspension 2 and 1 mg mL-1 for others; H2O2, 15 mM for suspension 2 and 0.75 mM for suspension 4; NaAuCl4, 5 mM. change slope as that without H2O2, indicating that the chemical oxidation treatment affected minor to the enzyme activity in this case. The good retainment of enzyme activity should be ascribed to that the oxidant H2O2 itself is a product of this kind of oxidase and the adopted concentration was also really low. Mass transfer efficiency in the biocomposites The mass transfer efficiency of MOCPsco was investigated and compared with conventional polymers. By transferring these polymers with identical BDT or DMcT content onto Au electrodes, we characterized and compared the mass transfer efficiencies by detecting the oxidation current of relevant electrodes at 0.7 V to H2O2 with identical bulk concentrations. Bare Au electrode was used as control. The current responses are shown in Figure 6(A) for BDT case and Figure 6(B) for DMcT case. For the BDT case, BDT-MOCPsco electrode showed current responses of 14 ± 0.9 µA, which is 1.8 and 7 folds of those in the BDT-MOCPs (8 ± 0.5 µA) and PBDT (2 ± 0.3 14


A

1

B

1

15 µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15 µA

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2

2

50 s

50 s

3

3

4

4

Figure 6. (A) The chronoamperometric responses to successive additions of 1 mM H2O2 obtained in PBS (pH 7.0) at 0.7 V vs. SCE for bare Au (1), BDT-MOCPsco (2), BDT-MOCPs (3), and PBDT-GOx (4) modified Au electrode. (B) The chronoamperometric responses to successive additions of 1 mM H2O2 obtained in PBS (pH 7.0) at 0.7 V vs. SCE for bare Au (1), DMcT-MOCPsco (2), DMcT-MOCPs (3), and PDMcT-GOx (4) modified Au electrode. µA) cases, respectively. This also reached 67% of that of bare electrodes (21 ± 1.4 µA). In the DMcT case, DMcT-MOCPsco electrode showed current responses of 16 ± 0.8 µA, which is 1.3 and 4.4 folds of those in the DMcT-MOCPs (12 ± 1.1 µA) and PDMcT (3.6 ± 0.7 µA) cases, respectively. This also reached 76% of that of bare electrodes. Note that the surficial properties of the unoccurpied area of the modified electrodes surface should be identical in BDT or DMcT case because all oligomers should have been cooridnated or washed away before the modification. Above results should solidly prove the superior mass transfer efficiency in the MOCPsco upon others. This merit should also greatly facilitate the biosensing, which is highly dependent on the mass transfer of substrates and products. The biosensing performance of different biocomposites Under optimized conditions (as listed in Table S1), the biosensing performance of BDT-MOCPsco- and DMcT-MOCPsco-modified Au electrodes was evaluated by detecting the amperometric response of H2O2 that is the product of the enzymatic catalysis of glucose. For comparison, polymers from chemical oxidation and metal-organic coordination on Au electrodes were also examined, as shown in Figures

15



400

400

A

1

5

0

j / µA cm-2

-2

j / µA cm

-2

2

0

100

y=135.42x+2.58 r2=0.9968

300

10

200

3

B

3 15

300

j / µA cm

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200

1

y=32.97x+4.56 2 r =0.9931

100

200 400 600

2

y=104.24x+2.09 r2=0.9978

t/s 0

0 0

200

400

600

800

1000

0

1200

2

4

6

8

cglucose / mM

t/s

Figure 7. The chronoamperometric responses to successive additions of glucose (A, the insert shows the responses at low concentrations of glucose) and the calibration curves (B) obtained in PBS (pH 7.0) at 0.7 V vs. SCE for PBDT-GOx (1), BDT-MOCPs (2), and BDT-MOCPsco (3) modified Au electrode fabricated under optimized conditions. The linearly regressed lines are also shown. 7 and S5, respectively. The biosensing sensitivity (S), linear response range, and limit of detection (LOD) are listed in Table 1. The sensitivities of the BDT-MOCPsco/Au electrodes was promoted to 135 µA cm-2 mM-1, which is 1.29 fold of that of the BDT-MOCPs/Au electrode (104 µA cm-2 mM-1) and 4.09 fold of that of the PBDT/Au electrode (33 µA cm-2 mM-1), respectively. The sensitivity of the DMcT-MOCPsco/Au electrodes was 104 µA cm-2 mM-1, which is 1.25 fold of that of the DMcT-MOCPs/Au electrode (83 µA cm-2 mM-1) and 4.95 fold of that of the PDMcT/Au electrode (21 µA cm-2 mM-1), respectively. Note that the sensitivities of BDT-MOCPsco/Au and DMcT-MOCPsco/Au electrodes are much better than that of most analogues.32-40 The LOD (S/N=3) are also two-magnitudes lower than those of reported analogues, as listed in Table S2. These sensitivities might also enter into the range of the most sensitive analogues.41 All results demonstrated the MOCPsco-based

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electrodes with notably improved biosensing performance comparing with those constructed by conventional polymer materials/strategies and most of reported analogues.

Table 1. Biosensing Performance of the Electrodes Modified with Different Biocomposites (r2 Values for LDR Are All >0.99) Modified Electrodes

Sensitivity (µA cm-2 mM-1)

LDR (µM)

LOD (nM)

BDT-MOCPsco/Au

135

1-1555

20

DMcT-MOCPsco/Au

104

10-1550

48

BDT-MOCPs/Au

104

1-1555

36

DMcT-MOCPs/Au

83

1-1555

65

PBDT/Au

33

10-4550

410

PDMcT/Au

21

10-50

630

The stability, selectively and feasibility for real sample detection of the biocomposites-based biosensors were investigated further. After 45-day storage in PBS at 4 oC, the BDT-MOCPsco/Au and DMcT-MOCPsco/Au electrodes remained 85% and 82% of initial response (Figure S6), demonstrating satisfactory stability. Additionally, the proposed enzyme composite-modified electrodes showed good selectivity to common interferences of uric acid and ascorbic acid (Figure S7). Furthermore, we evaluated the feasibility of the BDT-MOCPsco/Au electrodes to apply in blood serum sample, which reported biases of ±0.26 mM and recovery values of 94-104% via standard addition method (Table S3), indicating that the proposed enzyme electrode is promising in real sample detection. The outstanding performance

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of the MOCPsco-based enzyme electrodes should be ascribed to the benefits from the combined polymerization method with improved enzyme load, mass transfer efficiency and stability. CONCLUSIONS We have successfully explored a method combining the chemical oxidation polymerization for pre-lengthening ligands and the metal organic coordination polymerization for biosensing with significantly enhanced performance. This highly controllable pre-lengthening process could greatly enhance the encapsulation efficiency through forming active and protective oligomer shell, as well as increase the porosity of the polymers. This method significantly promoted the enzyme entrapping efficiencies, even near 100% in the BDT case, as well as much facilitated mass transfer efficiencies. The biosensing performance of proposed biocomposites was also greatly enhanced, comparing with conventional metal organic coordination enzyme biocomposites. The biocomposites modified electrodes were also promising for real detection based on the satisfactory stability and selectivity. This new materials/strategy may create new avenues to regulate the property of MOCPs and find broad applications in the fields of biosensing, biofuels, biocatalysis and environmental monitoring. ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (Grants 21105026, 21475041, 21505120), the Research Foundation of Education Bureau of Zhejiang Province (N20150189), the Opening Fund of Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), Hunan Normal University and the State Key Laboratory of Chemo/Biosensing and Chemometrics. Supporting Information Scheme for chemical oxidation of DMcT. Tables for performance of proposed biosensors and analogues, as well as assay results in blood samples. Figures for pictures and UV-vis spectra of chemical oxidation. Figures of the performance of 18


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TOC Graphic

2

1

Monomer

NaAuCl4 Path I

Oligomer

H2O2 3

Enzyme

Path II

4

MOCPs

NaAuCl4

23


Bioimmobilization Matrices with Ultrahigh Efficiency Based on

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