US-12618093-B2 - Method, an electrochemical sensor and a system for selective detection of infections

US12618093B2US 12618093 B2US12618093 B2US 12618093B2US-12618093-B2

Abstract

A method, an electrochemical sensor and a system for selective detection of infections. The method detects a concentration of nicotinamide adenine dinucleotide, NADH, from a bacterial culture through a cyclic voltammetry or chronoamperometry applied to an electrochemically active polymer. Therefore, prokaryotic cells can be detected while eukaryotic cells remain undetected. The infections can include bacterial infections and fungi or yeasts microbial infections.

Inventors

Brenda Guadalupe Molina García
Elaine Aparecida Armelin Diggroc
Pau TURON DOLS
Carlos Enrique ALEMÁN LLANSÓ

Assignees

UNIVERSITAT POLITECNICA DE CATALUNYA
B. BRAUN SURGICAL, SA

Dates

Publication Date: 20260505
Application Date: 20200311
Priority Date: 20190312

Claims (9)

1 . A method for selective detection of infections, the method comprising detecting a concentration of nicotinamide adenine dinucleotide (NADH), from a bacterial culture through a cyclic voltammetry or chronoamperometry applied to an electrochemically active polymer, wherein the infections include at least a bacterial infection; wherein the electrochemically active polymer comprises: particles of amphiphilic copolymers consisting of a polythiophene backbone with grafted biocompatible polycaprolactone and polyethylene glycol blocks, or a layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene) and a layer of poly(3,4-ethylenedioxythiophene), wherein the layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene) is deposited on top of a film of isotactic polypropylene and the layer of poly(3,4-ethylenedioxythiophene) is deposited on top of the layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene).
2 . The method according to claim 1 , wherein the electrochemically active polymer further comprises polythiophenes substituted at the 3-position of the thiophene ring.
3 . The method according to claim 1 , wherein the electrochemically active polymer comprises particles of amphiphilic copolymers consisting of a polythiophene backbone with grafted biocompatible polycaprolactone and polyethylene glycol blocks, and the electrochemically active polymer is deposited on top of or integrated in a non-toxic and biocompatible substrate.
4 . The method according to claim 3 , wherein the non-toxic and biocompatible substrate is made of polypropylene, polyesters, polyamides, polycarbonates, vitreous carbon, hydroxyapatite or a metal including platinum, gold, stainless steel, titanium, or magnesium alloys.
5 . A system for selective detection of infections, wherein the infections include at least a bacterial infection, the system comprising: a medical device; an electrochemical sensor including a solid substrate acting as a support, and an electrochemically active polymer deposited on top of said support and configured to be electrochemically activated, said electrochemical sensor, when in use, being adapted to be placed over a surface, part of the surface or in a body of said medical device; and a plurality of electrodes adapted and configured to apply an electrical potential to the electrochemically active polymer, wherein the medical device is configured to be located in a bacterial culture or located in a living tissue, and as a result of a cyclic voltammetry or chronoamperometry applied to the electrochemically active polymer, the electrochemically active polymer is configured to detect a concentration of nicotinamide adenine dinucleotide (NADH), and wherein the medical device comprises a suture, a surgical mesh, a vascular prosthesis, a hip prosthesis or a knee prosthesis wherein the electrochemically active polymer comprises: particles of amphiphilic copolymers consisting of a polythiophene backbone with grafted biocompatible polycaprolactone and polyethylene glycol blocks, or a layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene) and a layer of poly(3,4-ethylenedioxythiophene), wherein the layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene) is deposited on top of the solid substrate and the layer of poly(3,4-ethylenedioxythiophene) is deposited on top of the layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene), wherein the solid substrate is a film of isotactic polypropylene.
6 . The system according to claim 5 , wherein the plurality of electrodes includes screen printed electrodes (SPEs), or implantable electrodes.
7 . The system according to claim 5 , wherein the solid substrate is made of a non-toxic and biocompatible material including polypropylene, polyesters, polyamides, polycarbonates, vitreous carbon, hydroxyapatite or a metal including platinum, gold, stainless steel, titanium, or magnesium alloys.
8 . The system according to claim 5 , wherein the electrochemically active polymer comprises polythiophenes substituted at the 3-position of the thiophene ring.
9 . The system according to claim 5 , wherein: the medical device comprises the surgical mesh, the vascular prosthesis, or the hip prosthesis; and the solid substrate includes a film, a mesh, a suture or a three-dimensional device.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is the United States national phase entry of International Application No. PCT/EP2020/056487, filed Mar. 11, 2020, and claims the benefit of priority of German Application No. 19382178.2, filed Mar. 12, 2019. The contents of International Application No. PCT/EP2020/056487 and German Application No. 19382178.2 are incorporated by reference herein in their entireties. FIELD The present invention is directed, in general, to the field of electrochemical detection of prokaryotic cells. In particular, the invention relates to a method, to an electrochemical sensor and to a system for selective detection of bacteria, among other prokaryotic microbial agents such as fungi and yeasts. BACKGROUND Bacteria embed themselves in a hydrated extracellular matrix of polysaccharides and proteins, forming a slimy layer known as a biofilm. Biofilms, which are considered as an adaptation of microbes to hostile environments, are generated after initial adhesion of bacteria onto any kind of living or inert surface and their subsequent immobilization growth and reproduction. During the growth phase, bacteria produce extracellular biopolymers that extend developing a complex framework of molecular fibers with unique characteristics, the most important one being its capacity to hinder the access of antimicrobials through it. As a consequence, the adhered microorganisms increase their antimicrobial resistance, becoming up to one thousand times more resistant to antibiotics. In the biomedical field, bacterial biofilm infections, which are typically associated with patients with indwelling devices for the purpose of medical treatments, attract significant clinical investigations since once established, it becomes difficult to eradicate. Thus, with the progress of medical sciences, the application of medical prostheses and/or artificial organs in the treatment of human diseases is experiencing an exponential growth and, therefore, bacterial biofilm infections become more frequent. Unfortunately, the vast majority of internal (e.g. vascular prosthesis, cerebrospinal fluid shunts, prosthetic heart valves and breast implants) and external (e.g. dentures and contact lenses) prostheses, as well as hip prosthesis (i.e. when hip joints are replaced by prosthetic implants) may result in biofilm infections. Strategies for prevention of biofilm infections become therefore challenging and attract significant attention. One of the emerging methods for biofilm detection is the use of electrochemical impedance spectroscopy (EIS) measures employing polymeric sensors. In EIS, the electrochemical impedance across the electrode-electrolyte interface is carried out over a wide range of frequencies to elicit information about the properties of the interface. In the case of biofilms, successful detection is based on changes related to charge transfer resistance and capacitance corresponding to the maturing stages of biofilm development. However, analysis of the electrical behavior of the development of biofilms is not a simple task and development of simpler or more selective methods is highly desirable. Apart from that, eukaryotic cells present two major nicotinamide adenine dinucleotide (NAD) pools, the cytosolic and the mitochondrial pools. Although aerobic respiration reactions in eukaryotic cells take place in the mitochondria, the mitochondrial and cytosolic NAD ratio is cell-type specific. However, a distinctive characteristic of eukaryotic cells is that mitochondrial double-membrane is impermeable to reduced NAD (NADH) and oxidized NAD (NAD+), i.e. the outer membrane is quite permeable but the inner membrane is highly folded into cristae. As a consequence, the mitochondrial NAD levels are maintained even upon massive depletion of cytosolic NAD occurs. In opposition, as prokaryotes' do not have mitochondria, their whole respiration occurs in the cytosol or on the inner surfaces of the cells membrane. Therefore, as prokaryotic cellular membranes are permeable to NAD, the extracellular detection of NAD could be an appropriated approach for detecting the presence of bacteria in a eukaryotic cell environment, and as a result, to selectively identify growing bacterial infections on an implanted medical device. Thus, there is a need of: a) identify appropriated bacteria markers (i.e. chemicals with redox properties) for electrochemical detection related to microbial infections, and in particular of biofilm formation; and b) integrate electrochemical sensors for such bacteria markers into prosthetic and implantable materials. Several developments in this field have yielded electrochemical sensors for NADH. For example, in “Electrocatalytic oxidation of NADH at low overpotential using nanoporous poly(3,4)-ethylenedioxythiophene modified glassy carbon electrode”, Rajendran Rajaram et al. authors developed a conducting polymer (PEDOT) sensor for NADH against fouling. Unlike present invention, however, no