Recent Advances in Electrochemical Detection of Cell Energy Metabolism

Abstract

Cell energy metabolism is a complex and multifaceted process by which some of the most important nutrients, particularly glucose and other sugars, are transformed into energy. This complexity is a result of dynamic interactions between multiple components, including ions, metabolic intermediates, and products that arise from biochemical reactions, such as glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), the two main metabolic pathways that provide adenosine triphosphate (ATP), the main source of chemical energy driving various physiological activities. Impaired cell energy metabolism and perturbations or dysfunctions in associated metabolites are frequently implicated in numerous diseases, such as diabetes, cancer, and neurodegenerative and cardiovascular disorders. As a result, altered metabolites hold value as potential disease biomarkers. Electrochemical biosensors are attractive devices for the early diagnosis of many diseases and disorders based on biomarkers due to their advantages of efficiency, simplicity, low cost, high sensitivity, and high selectivity in the detection of anomalies in cellular energy metabolism, including key metabolites involved in glycolysis and mitochondrial processes, such as glucose, lactate, nicotinamide adenine dinucleotide (NADH), reactive oxygen species (ROS), glutamate, and ATP, both in vivo and in vitro. This paper offers a detailed examination of electrochemical biosensors for the detection of glycolytic and mitochondrial metabolites, along with their many applications in cell chips and wearable sensors.

Keywords: cell chip; electrochemical biosensor; glycolytic metabolites; mitochondrial metabolites; wearable sensor.

Figure 2

(A) Schematic illustration of the design of GOx@ZIF-8/CaCO₃ (GS NPs) and the flexible hydrogel-based biosensor. (B,C) EIS and CV graphs of different modified electrodes in 5 mM [Fe(CN)₆]^3−/4− containing 0.1 M KCl. Inset: Magnified view of EIS of bare Au electrode and the modified electrode, Au-TMSPMA. (D–F) Calibration curve of biosensor for glucose (Glc) vs. Glc concentration. EIS results of the biosensor response to Glc concentrations ranging from 0.25 to 4.0 mg/mL (Blue: EIS before Glc addition, Red: EIS after Glc addition). (G) The concept of smartphone-based glucose electrochemical detection platform (GEDP) integrated microneedle array (MA) with reverse iontophoresis (RI) technique for the minimally invasive detection of Glc in interstitial fluid (ISF). (H,I) The static current differences (ΔI) detected via the smartphone-based GEDP and the blood glucose levels (BGLs) measured by a commercial blood glucometer in healthy rats and diabetic rats. Dynamic chronoamperometric ΔI detected via same platform vs. BGLs measured in the tail vein blood using a commercial glucometer. (Reprinted with permission from [68]. Copyright 2022, Elsevier; reprinted with permission from [70]. Copyright 2022, Elsevier).

Figure 6

(A) Schematics of the surface of the working electrode in the process of the fabricated biosensor. Analytical performance of the GluBP-based biosensor. Cyclic voltammograms of SPCE, AuNP/SPCE, and GluBP/AuNP/SPCE in phosphate buffer (PB), and GluBP/AuNP/SPCE in the presence of 1 μM glutamate in PB. Scanning range is from −0.2 to +1.0 V vs. Ag/AgCl at a rate of 50 mV/s in 50 mM PB, pH 7.4. (B) Cyclic voltammograms of AuNP/SPCE in 50 mM PB (pH 7.4) and GluBP/AuNP/SPCE in the presence of different glutamate concentrations (0.0, 0.1, 0.5, 0.8, and 1.0 μM) in the same buffer. Inset: calibration plot for the change in the Au reduction peak current (I) vs. glutamate concentration. (C) Cross-reactivity of the GluBP-based sensor with other amino acids and ascorbic acid, a common interfering substance. (D) EIS characterization of SPCE, AuNP/SPCE and GluBP/AuNP/SPCE in 10 mM [Fe(CN)₆]^3−/4−. (E) X-ray crystal structure of GluBP35 demonstrating the binding pocket with charged amino acid residues stabilizing glutamate and neutral amino acid residues forming hydrogen bonds with glutamate. (F) The nanoaptasensors for the detection of ATP. (G) DPV responses of the nanoaptasensor to different concentrations of ATP in 10 mM PBS (pH 7.4), (a–h) 0, 0.05, 0.25, 0.50, 0.75, 1.0, 1.5, and 2.0 mM. Inset: calibration plot of ATP. (H) Histograms of the increased peak I’s of the nanoaptasensors for 1.0 mM ATP, 5.0 mM ATP analogues, 25 mM glucose, and cell culture medium. (I) Representative brightfield images of HeLa cells after nanoaptasensors inserted into different regions (nucleus, cytoplasm, and extracellular space). Scale bar: 20 μm. (J) I responses to insertion of the nanoaptasensor into different regions of five HeLa cells. Box graph summarizing the average ATP concentrations when nanoaptasensor inserted into different regions of five HeLa cells. (Reprinted with permission from [104]. Copyright 2021, Elsevier; reprinted with permission from [107]. Copyright 2020, American Chemical Society).

Figure 1

Schematic illustration of EC biosensors for cell metabolism detection and their applications.

Figure 3

(A) Schematic illustration of PANI film (above) and SEM images (below). (B) Amperograms (a) and calibration curve (b) of PANI-3/SPCE to 0.25 to 60 mM lactate in phosphate buffer; peak current (I) of PANI-3/SPCE to 10 mM interference substance and lactate (c); peak I of PANI-3/SPCE during 30 days storage (d); applied potential: −0.15 V; the reproducibility tests with five electrodes (e) and five consecutive (f) measurements of PANI-3/SPCE to 20 mM lactate. (C) Schematic diagram of the fabrication processes of the 3D LOx/PCP electrochemical sensor and collagen hydrogel integrated platform (above) and the mechanism of lactate detection on this platform (below). (D) Cyclic voltammetry (CV) for PBS recorded at PCP (red) and CNTs/PEDOT (black) electrodes. (E) CV results obtained at PCP electrode in PBS (pH 7.0) for 15 cycles. (F) CV results for PBS with (red) and without (black) 1 mM lactate at LOx/PCP electrode (scan rate, 10 mV/s). (G) Amperometric responses of LOx/PCP electrode to a series of increasing lactate concentrations in PBS solution at a potential at −0.05 V (vs. Ag/AgCl). Corresponding calibration curve of the electrode in lefthand panel. (H) Amperometric responses to 20 µM lactate and potential interfering agents at 100 µM concentration on proposed platform. (Reprinted with permission from [78]. Copyright 2022, Elsevier; reprinted with permission from [79]. Copyright 2022, Elsevier).

Figure 4

(A) Schematic diagram of NPQD modification (upper panel). Cyclic voltammetry (CV) data obtained during the electrochemical functionalization of the 4-aminothiophenol Au electrode in (a) 100 mM PBS (pH 7.4) and (b) 10 mM PBS (pH 7.4). The scan rate is 100 mV/s. (B) Electrochemical results for NADH in mouse serum. (a) Voltammogram results (potential range: from −100 to 700 mV). (b) CV from the circled part of graph (a). (C) (a) NADH quantification measured via CA. (b) Calibration plot of NADH in mouse serum (0–1000 µM). The current at 10 s in the CA graph was used for NADH quantification. (D) (a) CVs of bare SPE and PTZ/SPE in the absence and presence of 100 μM NADH. (b) PTZ/AgPNPs/SPE (c) PTZ/AgRNPs/SPE, and (d) PTZ/AgSNPs/SPE with sequential additions of each 20 μM NADH (20–100 μM). Insets of (b–d) are CVs of AgPNPs/SPE, AgRNPs/SPE, and AgSNPs/SPE in the absence and presence of 100 μM NADH. Electrolyte: 0.1 M phosphate buffer solution (pH 7.0) at a scan rate of 20 mV/s. (E) (a) CVs of PTZ/AgPNPs/SPE, PTZ/AgRNPs/SPE, and PTZ/AgSNPs/SPE for 50 continuous scans at the scan rate of 20 mV/s. (b,c) Long-term stability and reproducibility study of PTZ/AgRNPs/SPE in the absence and presence of 40 µM NADH. (d) Amperometric response of PTZ/AgRNPs/SPE to 15 µM NADH in the presence of 75 µM of various interfering species in 0.1 M phosphate buffer solution at an applied potential of +0.4 V. (Reprinted with permission from [94]. Copyright 2022, Springer Nature; reprinted with permission from [95]. Copyright 2021, Elsevier).

Figure 5

(A) Scheme of a 2D-Zn/Co-ZIF(HRP)|ZnCoO|Ti nanoarray biosensor for detecting H₂O₂ leakage from mitochondria after treatment with various inhibitors against the mitochondrial complexes. (B) Chronoamperometric results of increasing H₂O₂ concentration at (a) a 3D-Zn/Co-ZIF(HRP)|ZnCoO|Ti electrode. The corresponding calibration plots based on electrode surface area-normalized |steady-state current| in (a) in the low (b) and high (c) H₂O₂ concentration ranges. (d) A 2D-Zn/Co-ZIF(HRP)|ZnCoO|Ti. The corresponding calibration plots normalized |reduction peak current| in (d) in the low (e) and high (f) H₂O₂ concentration ranges. (g) Calibration plots based on |steady-state current| of HRP-modified GCE vs. H₂O₂ concentration. (h) Calibration plots of Zn/Co-ZIF|ZnCoO|Ti vs. H₂O₂ concentration. (i) Chronoamperometric responses of 2D-Zn/Co-ZIF(HRP)|ZnCoO|Ti obtained at −0.3 V via successive addition of H₂O₂ and various interfering agents. (C) Scheme of the synthesis of Ag/MNS-CeO₂-TiO₂ materials and the detection of cell-released superoxide anions (O₂^•−). (D) The i–t curve and bar graph (inset) of current (I) responses of MNS-CeO₂-TiO₂-450/SPCE, Ag/MNS-TiO₂/SPCE, and Ag/MNS-CeO₂-TiO₂-450/SPCE at −0.55 V in 0.1 M Ar-saturated PBS toward the addition of 0.08 mM O₂^•−. (E) EIS curves in 0.1 M KCl solution containing 5 mM Fe(CN)₆^3−/4−. (F) The i–t curve and bar graph (inset) of amperometric responses toward 0.08 mM O₂^•−, and the i–t curve and bar graph of amperometric responses toward 0.08 mM O₂^•− at each step in 0.1 M PBS (pH 7.4). (G,H) The amperometric responses and fitted linearity curve (inset) of Ag/MNS-CeO₂-TiO₂-450/SPCE toward different addition amounts of O₂^•− in 0.1 M PBS (pH 7.4) at applied potential of −0.55 V. (H) I responses toward 0.08 mM O₂^•−, 4 mM Glu, 0.4 mM UA, 0.08 mM AA, 0.08 mM DA, 0.08 mM AP, 0.14 M NaCl, 4 mM K₂SO₄, 0.08 mM H₂O₂, and O₂^•− added into 0.1 M PBS. (Reprinted with permission from [100]. Copyright 2023, Elsevier; reprinted with permission from [101]. Copyright 2021, Elsevier).

Figure 7

(A) Origin of redox signals in live cells and versatile applications. (B) CV signals obtained from isolated mitochondria, HeLa cells, and MMP-9. (C) DPV signals from HeLa cells treated with MMP inhibitors: 2 μM GKT136901, 2 μM Mito-Tempo (MT), 10 μM sodium diethyldithiocarbamate (DETC), and 10 μM batimastat (BB-94). Box plot of the calculated current (I) densities measured in the left panel (j indicates the I densities). (D) DPV signals with varying numbers of HeLa cells ranging from 7466 to 48,866 cells. Linear correlations (R²) of the calculated I densities from the DPV graph and final number of HeLa cells cultured on the HCGN platform. (E) DPV signals (E_p = −0.02 V) elicited from isolated mitochondria with various numbers of cultured HeLa cells. Linear correlations (R²) of the calculated I densities from the DPV graph and the mitochondria isolated from HeLa cells. (F) Schematic illustration and photos of the HIS paper. (G) The sensor properties of the HIS paper. (a) Amperometric response and calibration curve of the glucose (Glc) sensor with Glc concentrations of 0, 0.075, 0.15, 0.25, 0.45, 0.65, 0.95, and 1.25 mM in 60 μL of 0.1 M PBS (pH 6.71), −0.18 V. Inset: the calibration curves of Glc. (b) Interference study in the presence of 0.15 mM Glc (plot c), followed by subsequent 50 μM additions of lactate (plot a), uric acid (plot b), and ascorbic acid (plot d). (c) Reproducibility of the Glc sensors manufactured in different batches at Glc concentrations of 0.15, 0.25, and 0.45 mM. (d) Amperometric response and calibration curve of the lactate sensor with lactate concentrations of 0, 0.3, 2.3, 5.3, 8.3, 12.3, 16.3, and 20.3 mM in 60 μL of 0.1 M PBS (pH 6.71), −0.20 V. Inset: the calibration curves of lactate. (e) Interference study in the presence of 2.0 mM lactate (plot a), followed by subsequent 50 μM additions of uric acid (plot b), Glc (plot c), and ascorbic acid (plot d). (f) Reproducibility of the lactate sensors manufactured in different batches at lactate concentrations of 2.3, 5.3, and 8.3 mM. (Reprinted with permission from [115]. Copyright 2023, Wiley; reprinted with permission from [116]. Copyright 2021, Elsevier).