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Systems and methods for detecting and monitoring proteolysis of protein matrices

A protein matrix and proteolysis technology, applied in the field of analytical instruments for electrochemical testing, can solve the problems of ineffective, expensive, and unsuitable for point-of-care or on-site diagnosis of fibrinolysis in timely and sensitive detection and monitoring

Inactive Publication Date: 2019-03-26
DIGITAL DIAGNOSTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, tests to detect or monitor fibrinolysis are usually limited to sophisticated methods in specialized laboratories and to native fibrin clots, such as thromboelastometry (TEM), and are not suitable for point-of-care or point-of-care diagnostics
Other methods, using colorimetric or fluorometric assays, are also expensive, complex, and negatively affected by the color or turbidity of the sample, and thus are not effective for timely and sensitive detection and monitoring of fibrinolysis

Method used

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  • Systems and methods for detecting and monitoring proteolysis of protein matrices
  • Systems and methods for detecting and monitoring proteolysis of protein matrices
  • Systems and methods for detecting and monitoring proteolysis of protein matrices

Examples

Experimental program
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Effect test

Embodiment 1

[0153] Example 1: Voltammetry of proteolysis

[0154] Mix human fibrinogen and human thrombin stock solutions to produce 10 μl of final solution (2% fibrinogen + 0.1 U thrombin final concentration), vortex and immediately apply to 5x5 filter paper strips (Whatman No.4 , with a large pore size, retaining 30-40 micron particles). Fibrin clots formed within the pores of the strips and the strips were allowed to dry for 2 hours at room temperature. Due to the fibrinogen concentration, the clot pores are smaller than the pores in the filter paper.

[0155] With 50mM K containing 0.2% Tween20 3 Fe(CN) 6 The solution impregnates the paper strip-clot and allows it to dry. Stick the paper strips to the chip containing lithographically printed electrodes (gold or conductive carbon). An optional silver wire can be attached to the paper strip as a reference electrode ( Figure 3C ). The strip was rehydrated with phosphate-buffered saline (PBS) containing 2 nM human plasmin. PBS wi...

Embodiment 2

[0156] Example 2: CES and WES for Chronoamperometry

[0157] Reference is made to the above mixed composition of WES and CES for chronoamperometry measurements. For example, in the Fc-PEG electromotive discussed above, the electroactive Fc moiety has a standard electrode potential of +0.641 V, and the electromotive itself has an even higher E0 (measured E0 in the +0.7V to 0.75V range). If the reduced form of WES is used for measurements at WE, this high oxidation resistance - relative to other commonly used electroactive species - provides resistance to many oxidized CESs. However, as discussed above, due to the excess concentration of CES, it is advantageous to use a weak oxidizing agent as CES so that WES exists mainly in its reduced state.

[0158] Correspondingly, for example, a suitable CES is hexamineruthenium(III)Ru(NH 3 ) 6 3+ , which has a standard electrode potential E0 of +0.1V. Based on the Nernst equation, containing 2mM reduced PEG-Fc WES and 8mM Ru(NH 3 )...

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Abstract

Devices and methods are provided for detecting and monitoring proteolysis of protein matrices, such as fibrin clots, extracellular matrices, and collagen matrices. The apparatus and methods generally include measuring diffusion-limited electrochemical currents generated by electroactive species or elactomers in a synthetic protein matrix. The devices and methods are in various embodiments used to detect and monitor the proteolytic activity of a sample, and more specifically, the fibrinolytic activity and collagenase activity of a sample. The devices and methods are generally used to monitor and diagnose cardiovascular, cerebrovascular and tumor conditions.

Description

technical field [0001] The present disclosure generally relates to analytical instruments and methods for electrochemical testing. The present disclosure specifically relates to electrochemical detection of proteolysis of protein matrices. More specifically, novel devices and methods are provided for detecting proteolysis of protein matrices, including fibrin clots, extracellular matrix, and similar collagen or related protein-based matrices, whether endogenous or synthetic. Background technique [0002] Protein matrices are important for tissue function, tissue regeneration, wound healing, and hemostasis. For example, many eukaryotic cells are encapsulated by an extracellular matrix of proteins that provide the cell with structural support, cell and tissue determination, and autocrine, paracrine, and juxtrine properties, and are therefore necessary for normal cellular function . In wound healing, a cascade of molecular and cellular events initially leads to hemostasis—pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/416
Inventor K.迪米特洛夫
Owner DIGITAL DIAGNOSTICS INC
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