Label-free multiplexed electrochemical immunosensor for early diagnosis of lysosomal storage disorders – Scientific Reports

Lysosomal storage disorders (LSDs) are a group of inherited genetic diseases that cause fat accumulation in tissues and cells.1caused by mutations in the genes that encode enzymes inside the body2,3,4. All LSDs share the same property that causes the accumulation of naturally degraded substrates in lysosomes5It causes an accumulation of substrates resulting in cascading cell destruction and disruption, leading to tissue dysfunction.2,6. The severity of LSDs depends on the nature and amount of substrate accumulated6. LSD patients appear normal at birth, but develop symptoms early in childhood7. Neurological symptoms include brainstem dysfunction and seizures, peripheral symptoms include kidney and heart injuries, muscular atrophy, eye disease, enlarged liver and spleen, and irregular bone growth8. However, treatment is available for most LSDs if detected early in childhood, such as enzyme replacement therapy (ERT), bone marrow or stem cell transplantation and gene therapy, so early detection of LSD is critical.6,7.

Pompe disease (glycogen storage disease type II) is LSD caused by a deficiency of the enzyme lysosomal alpha-glucosidase (GAA). GAA is essential in the breakdown of glycogen into glucose and thus its deficiency leads to the accumulation of glycogen in organelles9,10. As a treatment for Pomp, patients are given an alpha-glucosidase enzyme such as ERT, which breaks down glycogen into glucose to reduce its accumulation in cells. However, alpha-glucosidase therapy should be started as soon as possible in infants.11. Another LSD is Krabbe disease (KD) or globular cell leukodystrophy (GCL), a neurological condition caused by galactocerebrosidase (GALC) deficiency. GALC is essential for the degradation of galactosylceramide in the white matter of the cerebrospinal nervous system. Clinically visible within 6 months of birth and death within 24 months if left untreated.12, 13. Gaucher disease is the most common type of sphingolipoidosis, caused by a deficiency of β-glucocerebrosidase (GBA)14It causes the accumulation of glucosylceramide in macrophages15thand are categorized into three main subtypes based on the presence or absence of neurological involvement16. Among all LSD diseases, Pompe, Gaucher and Krabi diseases are the most common and have severe symptoms that end in death. However, these diseases have available treatments and can be detected in childhood. Thus, if it is detected and diagnosed early in the infancy of the patient, treatment can be given accordingly.

It has been shown that the initial identification of patients with LSD can be achieved by immunofluorescence measurement of enzymes and lysosomal proteins, in which mutations lead not only to decreased enzyme activity, but also to a decrease in the amount of protein7. Deficiency patients have protein levels below the final concentration of about 3-100 ng/ml in healthy individuals17. Previously used methods have been reported for the quantification of some LSD-related proteins such as fluorescence, tandem mass spectrometry (LC-MS/MS) and immunoassay such as enzyme-linked immunosorbent assay (ELISA)2,7. However, these methods are known to be time-consuming, time-consuming to analyze, expensive, require specialized laboratories, and require a large sample volume, making them not ideal for point-of-care testing (POCT).18. Point-of-care testing enables quick access to results and thus provides faster monitoring, treatment selection, diagnosis and prognosis for diseases, leading to better decisions that are often vital to a patient’s health. To achieve the most effective methods of point-of-care testing, more rapid and cost-effective methods are being developed19.

In order to speed up and facilitate clinical diagnosis and testing of tests at the point of care, multiple analysis of different biomarkers produces faster and more accurate results. Thus, multiplex analysis using a single analytical device holds great promise in upgrading and simplifying diagnostic procedures, providing more data faster. Multiplexed detection offers several advantages such as using a lower sample size, lower average analysis time, more statistically reliable inferences and more informative detection results.20.

Biosensors are developing as interesting, cheaper, simpler and more sensitive alternatives to traditional methods of detection for diagnostic purposes. More specifically, immunoelectrochemical sensors are extensively researched in biomedical research and diagnostics, due to their high sensitivity, rapid response, reduced sample size used, miniaturization ability, and multiplexing capacity. Electrodeposition of gold nanoparticles (AuNPs) electrochemical immunosensors via temporal mixing measurement, improves their performance by enhancing the electron transfer rate and catalytic activity of the sensor.21. This is done by reducing HAuCl4 using potassium nitrate. AuNPs increase the surface area allowing more antibodies to be attached to the surface of the transducer resulting in significantly higher signal and sensitivity. The electrodes modified with gold nanoparticles showed a nearly threefold increase in the area of ​​electrical activity, which increased the functional density of the biomolecules as well as improved electron exchange and sensitivity.22,23,24.

In this work, we report a new multiplexed electrochemical immunosensor developed for the simultaneous quantification and detection of GAA, GBA and GALC. Disposable carbon micro-flakes electrostatically deposited with AuNPs were used due to their high conductivity and high surface area allowing more antibody to be stabilized. GAA, GBA, and GALC antibodies were immobilized on the sensor to detect proteins. This multiplexed sensor can be used in the critical early diagnosis of LSDs in newborns in order to administer the correct treatment.

2022-06-04 09:37:30

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