Amperometric Biosensor of Matrix Metalloproteinase-7 Enhanced by Pd-Functionalized Carbon Nanocomposites
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Matrix metalloproteinase-7 plays a pivotal role in tumour progression and metastasis as an enzyme that can degrade the cell-matrix composition and cleave peptides between alanine and leucine in various biomolecular activation processes. In this work, a Pd-functionalised carbon nanocomposite was designed as a new impedance enhancer for an amperometric sensor of MMP-7. Pd nanoparticles in the enhancer can catalyse the oxidation of 4-chloro-1-naphthol with H2O2 to generate insoluble precipitation in situ, forming high-resistance precipitation on electrodes. In addition, poorly conductive carbon nanospheres of the nanocomposite increased the precipitation resistance, further causing a dramatic increase in resistivity of the enhancer and, subsequently, a significant decrease in current. This can significantly promote the current signal difference between the biosensor treated with and without the target analyte, which is directly related to the sensitivity of the amperometric biosensor. Overall, electrochemical biosensor can sensitively detect MMP-7 in the range of 100 fg mL−1 to 100 ng mL−1 with a limit of detection for MMP-7 of 17.38 fg mL−1.
KeywordsMatrix metalloproteinase-7 Tumour marker Peptide cleavage Impedance enhancer Amperometric biosensor
Bovine serum albumin
Energy dispersive X-ray spectroscopy
Fast Fourier transformation
Glassy carbon electrode
High-resolution transmission electron microscope
Limit of detection
Neuron specific enolase
Phosphate buffer solution
Pd-functionalized carbon nanocomposites
Relative standard deviations
Scanning electron microscope
Square wave voltammetry
Thrombin of bovine serum
Transmission electron microscope
Matrix metalloproteinase-7 (MMP-7), an enzyme that can degrade the composition of extracellular matrix , is highlighted for its pivotal role in tumour progression and metastasis [2, 3]. The content of MMP-7 in serum samples is associated with lymph node metastasis in patients with some cancers, such as salivary gland cancer , colon adenocarcinoma  and high-grade renal cell carcinoma . Due to its various roles in physiological processes, high sensitive and accurate detection of MMP-7 has attracted intensive research attention , leading to the development of several approaches, including colorimetry , electrochemiluminescence (ECL)  and fluorescence resonance energy transfer (FRET) analysis . Nevertheless, the limit of detection (LOD) of these approaches is typically in the picogram range and, thus, not low enough. Compared to these methods, electrochemical biosensors offer much more advanced MMP-7 detection capacities with lower LOD in femtogram level . Moreover, some analytic methods have been constructed to meet the urgent requirement for ultrasensitive electrochemical detection of MMP-7 using electrochemical assays due to their low cost and miniaturisation .
In many electrochemical protocols, enzymatic biocatalytic reaction is applicable in signal amplification to promote the performance of the amperometric biosensor [13, 14]. However, it was well known that enzyme, the most widely used catalyst in catalytic reactions, had obvious shortcomings in both environmental-sensitive activity and low stabilities [15, 16, 17, 18, 19]. Therefore, developing a high efficient and stable catalyst is a top priority in constructing an ultrasensitive electrochemical assay for MMP-7 detection. Pd is a noble metal material with superior catalytic properties and possesses high chemical stability in catalytic reactions [20, 21]. In addition, carbon material can act as a chemical inert support in catalysts to adsorb noble metal materials and retain catalytic properties [22, 23].
Considering above situations, we designed a Pd-functionalised carbon nanocomposite as an impedance enhancer to dramatically increase the sensitivity of an amperometric assay of MMP-7, which has the following two functions. (1) Carbon nanospheres are a poor-conductive material ; (2) Pd nanoparticles can catalyse the oxidation of 4-chloro-1-naphthol with H2O2 to generate insoluble precipitation in situ, forming high-resistance precipitation on electrodes . These two factors increase resistivity and significantly reduce current, which can remarkably improve the sensitivity of the biosensor to have a low LOD of 17.38 fg mL−1. The constructed Pd-functionalized nanocomposite for catalytic precipitation reaction is practical in amperometric assays of MMP-7 with high selectivity and sensitivity.
HAuCl4·3H2O, H2PtCl4, 4-CN, glucose, H2O2 (30%), thrombin of bovine serum (TBS) were purchased from Alfa Aesar (Tianjin, China). Graphene oxide (GO) was purchased from JCNANO (Nanjing, China). Bovine serum albumin (BSA, standard grade) was commercially achieved from Beijing Xinjingke Biotechnologies Co., Ltd. (Beijing, China). The peptide (NH2-KKKRPLALWRSCCC-SH) was obtained from Science Peptide Biological Technology Co., Ltd. (Shanghai, China). Neuron-specific enolase (NSE) and prostate-specific antigen (PSA) were purchased from Shanghai Linc-Bio Science Co. Ltd. (Shanghai). Matrix metalloproteinase-2 (MMP-2) was purchased from Yeasen Biotechnologies Co., Ltd. (Shanghai, China). MMP-7 was provided from Sino Biological Inc. (Beijing, China). Clinical human serum samples were purchased by ZhongKe ChenYu Biotech (Beijing, China). All aqueous solutions were prepared with ultrapure water (resistivity > 18 MΩ cm). The phosphate buffer solution (PBS) contains 0.1 M KCl and 10 mM phosphate buffer (pH = 7.4).
The microwave synthesis was carried out through CEM Discover® SP Microwave reactor (CEM, USA). Scanning electron microscope (SEM) images were obtained by using HITACHI S-4800 SEM (HITACHI, Japan). Transmission electron microscope (TEM) images were obtained on HITACHI H7650 TEM (HITACHI, Japan). Energy dispersive X-ray spectroscopy (EDS) was determined on HITACHI SU8010 SEM (HITACHI, Japan). All electrochemical measurements were carried out on CHI600 electrochemical workstation (Chenhua Instruments Co., Shanghai, China). A glassy carbon electrode (GCE) (4 mm in diameter) was used as working electrode, a platinum wire and a Ag/AgCl electrode were as counter electrode and reference electrode, thus a three electrochemical system in experiment was constructed. High-resolution transmission electron microscope (HR-TEM) images were performed by Tecnai G2F30 TEM under 300 kV accelerating.
Synthesis of the Pd-Functionalized Carbon Nanocomposites
Pd-functionalized carbon nanocomposites (Pd-CNCs) were synthesised according to a previously reported literature . Briefly, 4 g of glucose was dissolved into 40 mL of ultrapure water to form a clear solution. The above solution was then transferred into a 50 mL Teflon-lined stainless-steel autoclave and kept at 170 °C for 5 h. After reaction, the transparent solution turned to dark brown. The obtained carbon nanospheres were collected via centrifugation and washed by ultrapure water/ethanol for several times. The resulted products were redisposed in 8 mL of ultrapure water.
To functionalize Pd nanoparticles on carbon nanospheres , 1 mL carbon nanospheres suspension was mixed with 25 μL HPdCl4 solution. The mixture was reacted in microwave reaction instrument (250 W) at 100 °C for 15 min and then cooled down to the room temperature naturally. Next, the obtained Pd-carbon nanospheres were centrifuged, washed by ultrapure water and redisposed in 1 mL of ultrapure water.
To avoid the unspecific adsorption of MMP-7, BSA was further modified on the Pd-carbon nanospheres. The nanospheres suspension was stirred with 100 μL BSA solution (1 wt%) for 1 h under room temperature. After several centrifugation and washing steps, the BSA-modified Pd-carbon nanospheres were redisposed in ultrapure water and stored in 4 °C for further experiments.
Electrodeposition Au-rGO on GCE
Before modification, the GCE was polished with 0.05 μm alumina slurry and sonication washed in ultrapure water and ethanol, respectively. The electrodepositing solution was configured by the following steps . Firstly, 8 mg GO powder was dispersed in 20 mL ultrapure water under sonication for 2 h. Then, 200 μL HAuCl4 solution (4 wt%) was added into the GO suspension Subsequently, the electrodeposition of Au-rGO on GCE by cyclic voltammetry (CV) technique in the range between 1.5 and − 1.5 V with the scan rate of 50 mV s−1 in the above electrodepositing solution. Finally, the Au-rGO deposited GCE (Au-rGO/GCE) was washed by ultrapure water to remove the residual electrodepositing solution and then blow dried by nitrogen at room temperature.
Fabrication of the Biosensor
The Au-rGO/GCE was incubated with 40 μL peptide solution (50 μM) for 40 min at 37 °C. Subsequently, the modified peptide on the GCE was activated with 50 μL glutaraldehyde solution (0.10 wt%) for another 30 min (peptides/Au-rGO/GCE). Then, 20 μL Pd-CNCs suspension was dropped on the peptide-modified electrode for 1 h (Pd-CNCs/peptides/Au-rGO/GCE). After each modification step, the modified electrode was washed by ultrapure water.
Eighty microliters of MMP-7 solution (1 ng mL−1) was incubated with the Pd-CNCs/peptides/Au-rGO/GCE for 1 h at 37 °C and sufficiently washed by ultrapure water. Then, 50 μL of 1.0 mM 4-CN solution containing 10 mM H2O2 was dropped to proceed precipitation reaction for 50 min. Finally, the square wave voltammetry (SWV) measurement was conducted from − 0.2 to 0.6 V in 5 mM [Fe(CN)6]3−/4− phosphate-buffered solution (0.1 M, pH = 7.4) with pulse amplitude of 25 mV and an increase potential of 4 mV s−1.
Results and Discussion
Principle of the Peptide Cleavage Biosensor
Characterisation of the Pd-CNCs
Characterisation of Construction Procedures of the Biosensor
Optimization of Detection Conditions
Performance of Proposed Biosensor
Comparison of some recently reported biosensor for detection of MMP-7
Material and method
DNA enzyme-decorated DNA nanoladders
Differential pulse voltammetry
0.05 pg mL−1
2 × 10− 4–20 ng mL−1
Kou et al. 2016 
Fluorescence resonance energy transfer
0.22 pg mL−1
1 × 10− 2–100 ng mL−1
Lei et al. 2017 
0.1 μg mL−1
0–2 μg mL−1
Chen et al. 2013 
Magnetic peptide−DNA probe
Square wave voltammetry
0.02 pg mL−1
1 × 10− 4–50 ng mL−1
Wang et al. 2017 
Square wave voltammetry
3.1 fg mL−1
1 × 10− 5–10 ng mL−1
Zheng et al. 2018 
Square wave voltammetry
17.38 fg mL−1
1 × 10− 4–100 ng mL−1
Evaluation of Performance of Immunosensor
Recovery tests for MMP-7 in human serum samples
Added (ng mL−1)
Detected (ng mL−1)
Average recovery (%)
In summary, a Pd-functionalized carbon nanocomposite was fabricated as a novel impedance enhancer, which reveals promising H2O2 catalytic performance for 4-CN oxidation. Utilising the high impedance of insoluble oxidised 4-CN precipitation on the electrode, an amperometric biosensor for detection of MMP-7 was constructed. The biosensor possesses comparable sensitivity, a broad detection range, good practicability and outstanding selectivity to the detection of MMP-7, which suggests it potential application in various bio-applications. Our work further highlights the importance of the impedance enhancer in improving the performance of amperometric assays, encouraging the fabrication of new enhancers with advanced catalytic activity and high resistance.
This research was financed by grants from the National Natural Science Foundation of China (21673143, 21273153), Natural Science Foundation of Beijing Municipality (2172016, 2132008), High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (IDHT20180517) and Capacity Building for Sci-Tech Innovation—Fundamental Scientific Research Funds (025185305000/195).
Availability of Data and Materials
All data are fully available without restriction.
ZW carried out the experiments and drafted the manuscript. HQW designed the measurement study. ZFM and HLH supervised the overall study and polished the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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