Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor

doi:10.1016/j.ab.2004.05.047

Analytical Biochemistry

Volume 332, Issue 1, 1 September 2004, Pages 160-167

https://doi.org/10.1016/j.ab.2004.05.047 Get rights and content

Abstract

We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20 nm and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5 pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1 pM MMP-2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations.

Section snippets

Surface plasmon resonance measurements

Binding experiments were done with the SPR-based instrument Biacore 2000 and sensor chips CM3 and CM5 using the control software version 2.1 and evaluation software version 3.0 (Biacore AB, Uppsala, Sweden). CM3 chips were used for the one-step sandwich assays, CM5 chips were used for ligand characterization.

The running buffer contained 7.8 mM NaH₂PO₄, 8 mM Na₂HPO₄, 137 mM NaCl, 0.1 mM CaCl₂, 3 mM KCl, 1.5 mM KH₂PO₄ and 0.02% (v/v) Tween 20, pH 7.2 [4]. This buffer was also used for sample dilution.

Selection and characterization of two ligands for the development of a MMP-2 sandwich assay

To build up a sandwich assay for MMP-2 it was necessary to find two ligands recognizing different sites on the MMP-2 molecule. Three ligands were investigated with Biacore 2000 in detail—gelatine (substrate), anti-MMP-2 antibody Ab-2, and TIMP-2 (inhibitor). Binding of the ligands to MMP-2 was tested in two formats, with immobilized ligand proteins (Table 2, Fig. 1) and in the reverse format with immobilized MMP-2 (Table 3, Fig. 2). The binding response was read 10 s after the beginning of the

Discussion

A highly sensitive assay has been developed by combination of SPR with a particle-enhanced sandwich assay which allows the detection of subpicomolar concentrations of human MMP-2.

Using the proform of MMP-2 for calibration, the lower detection limit was 0.5 pM MMP-2. Since both TIMP-2 [4], [5] and the commercially available antibody Ab-2 bind both the proform and the active form of MMP-2, the assay should be applicable for the determination of both forms of MMP-2.

While optimizing the assay

Acknowledgements

The authors thank Dr. Thorsten Stroh for helpful discussions.

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Atomically-tailored graphene oxide displaying enhanced fluorescence for the improved optical sensing of MMP-2
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Modulation of the optical properties of two-dimensional nanomaterials is a crucial step to improve their sensing performance for the optical detection of target molecules of interest. Here, we report a mild, simple and effective approach for heteroatoms-doping of graphene oxide (GO) without size alteration, resulting in enhanced fluorescence properties. Sulfur and nitrogen atoms were chemically doped onto the basal plane of GO (NSGO) via a sonic wave-assisted reaction with cysteine as an S and N dopant under acidic conditions at room temperature. As-prepared NSGO retained the same lateral size as pristine GO, but possessed a large proportion of thiopyran sulfur and pyrrolic nitrogen. In addition, NSGO exhibited highly enhanced fluorescence with strong resistance to the pH and polarity of solution. Mechanistic investigation suggested that S-doping was essential for the improved fluorescence properties of NSGO. Finally, to investigate the effect of heteroatoms-doping on sensing performance, fluorescent NSGO was employed as a fluorescent biosensor for the detection of matrix metalloproteinases 2 (MMP-2) via a peptide-induced assembly and self-quenching principle. As compared to pristine GO, NSGO assemblies exhibited more significant fluorescence quenching, but upon addition of MMP-2, the NSGO assemblies were disassembled, leading to the higher restoration of the quenched fluorescence, eventually resulting in the more sensitive detection of MMP-2.
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^☆: Financial support by the Federal Ministry of Education and Research, Germany (InnoRegio-BioHyTec Project No. 03i1304C) is gratefully acknowledged.

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Analytical Biochemistry

Abstract

Section snippets

Surface plasmon resonance measurements

Selection and characterization of two ligands for the development of a MMP-2 sandwich assay

Discussion

Acknowledgements

References (26)

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 Å resolution

J. Mol. Biol.

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2

J. Biol. Chem.

Identification of the tissue inhibitor of metalloproteinases-2 (TIMP-2) binding site on the hemopexin carboxyl domain of human gelatinase a by site-directed mutagenesis

J. Biol. Chem.

Matrix metalloproteinases in tumor invasion and metastasis

Semin. Cancer Biol.

Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor

Biosens. Bioelectron.

The resonant mirror: a novel optical sensor for direct sensing of biomolecular interactions Part II: Applications

Biosens. Bioelectron.

The bidiffractive grating coupler: application to immunosensing

Sens. Actuators B

Highly sensitive optical chip immunoassays in human serum

Biosens. Bioelectron.

Optical chip immunoassay for hCG in human whole blood

Biosens. Bioelectron.

Quantitative zymography: detection of picogram quantities of gelatinases

Anal. Biochem.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases, structure, function, and biochemistry

Circ. Res.

E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

Biochemistry

Cited by (39)

Target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 activity

Construction of a sensitive protease sensor with DNA-peptide conjugates for single-molecule detection of multiple matrix metalloproteinases

Atomically-tailored graphene oxide displaying enhanced fluorescence for the improved optical sensing of MMP-2

Biosensors for measuring matrix metalloproteinases: An emerging research field

Surface plasmon resonance based biosensor for discovery of new matrix metalloproteinase-9 inhibitors

Development of a label-free SPR sensor for detection of matrixmetalloproteinase-9 by antibody immobilization on carboxymethyldextran chip

Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor☆

Abstract

Section snippets

Surface plasmon resonance measurements

Selection and characterization of two ligands for the development of a MMP-2 sandwich assay

Discussion

Acknowledgements

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Semin. Cancer Biol.

Biosens. Bioelectron.

Biosens. Bioelectron.

Sens. Actuators B

Biosens. Bioelectron.

Biosens. Bioelectron.

Anal. Biochem.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases, structure, function, and biochemistry

Circ. Res.

E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

Biochemistry