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Label-free multiplex electrochemical immunosensor for early diagnosis of lysosomal storage disorders

Lysosomal storage disorders (LSDs) are a group of inherited genetic diseases that cause lipids to accumulate in tissues and cells1resulting from mutations in genes encoding intralysosomal enzymes2,3,4. All LSDs share the same characteristic that they cause accumulation of naturally degraded substrates in lysosomes5they cause the accumulation of substrates leading to destruction and disfunction of cells, consecutively, causing tissue disfunction2.6. The severity of the LSDs depends on the nature and quantity of the accumulated substrate6. LSD patients appear normal at birth, but they develop symptoms early in childhood7. Neurological symptoms include brainstem dysfunction and seizures, and peripheral symptoms include kidney and heart injuries, muscle atrophy, ophthalmic diseases, enlargement of liver and spleen and irregular bone development8. However, treatment is available for most LSDs if discovered early in the infantile stage, such as enzyme replacement therapy (ERT), bone marrow or stem cell transplantation and gene therapy, therefore early detection of LSDs is crucial6.7.

Pompe disease (glycogen storage disease type II) is an LSD caused by the deficiency of the lysosomal enzyme acid α-glucosidase (GAA). GAA is necessary in degrading glycogen to glucose thus its deficiency leads to the accumulation of glycogen in organelles9.10. As treatment for pompe, acid α-glucosidase enzyme is given to patients as ERT, it breaks down glycogen to glucose to reduce its buildup in cells, however, treatment with acid α-glucosidase should be started as early as possible in infantseleven. Another LSD is Krabbe disease (KD) or Globoid cell leukodystrophy (GCL), is a neurological condition caused by the deficiency of galactocerebrosidase (GALC). GALC enzyme is essential for the degradation of galactosylceramide in the white matter of the cerebrospinal nervous system. Krabbe becomes clinically apparent within 6 months of birth and ends in death within 24 months if not treated12,13. Gaucher disease is the most common sphingolipidosis, it results from the deficiency in β-Glucocerebrosidase (GBA)14it causes the accumulation of glucosylceramide in macrophagesfifteenand it is classified into three main subtypes based on the presence or absence of neurological involvement16. Among all LSDs, Pompe, Gaucher and Krabbe diseases are the most common and have severe symptoms that end with mortality. However, these diseases have treatments available, and are detectable in their infantile stage. Hence, if detected and diagnosed early in the patient’s infantile stage, treatments can be administered accordingly.

It was proven that the initial identification of LSD patients can be achievable by immunoquantification of lysosomal enzymes and proteins, since mutations lead to not only deficiency in the enzyme activity but also cause diminishment in the amount of protein7. Deficient patients have protein levels lower than the cut-off concentration which is about 3–100 ng/ml in healthy individuals17. Previously used methods for the quantification of some LSD related proteins were reported such as fluorescence, tandem mass spectrometry (LC–MS/MS) and immunoassays like enzyme linked immunosorbent assay (ELISA)2.7. Nonetheless, these methods are known to be time consuming and take long analysis time, costly, require specialized laboratories and require large sample volume, making them not ideal for point-of-care testing (POCT)18. POCT allows rapid access to results therefore providing faster monitoring, choice of treatment, prognosis and diagnosis of diseases, resulting in better decision making which is often vital to patient’s health. To achieve POCT in the most effective way, methods that are more cost-effective and rapid are being developed19.

In order to expedite and facilitate clinical diagnosis and POCT, multiple analysis of different biomarkers produces faster and more accurate results. Thus, multiplexed analysis utilizing a single analytical device holds a great promise in upgrading and simplifying diagnostic procedures, as it provides more data, quicker. Multiplexed detection provides numerous advantages such as utilizing less sample volume, less averaged analysis time, more statistically reliable conclusions and more informative detection outcomes.twenty.

Biosensors are evolving to be an interesting cheaper, simpler and more sensitive alternatives for conventional methods of detection for diagnostic purposes. More specifically, electrochemical immunosensors are being researched extensively in the field of biomedical research and diagnostics, due to their high sensitivity, rapid response, minimization of sample volume used, their capability of being miniaturized and their ability of multiplexing. Electrodepositing electrochemical immunosensors with gold nanoparticles (AuNPs) via chronoamperometry, improves its performance via the enhancement of electron transfer rate and catalytic activity of the sensortwenty-one. It is done by reducing HAuCl4 using potassium nitrate. AuNPs increase the surface area allowing more antibodies to immobilize to the surface of the transducer resulting in a significantly higher signal and sensitivity. Gold nanoparticle-modified electrodes demonstrated a nearly threefold increase in electroactive area, resulting in an increase in functional density of biomolecules as well as improved electron exchange and sensitivity22,23,24.

In this work, we report a novel multiplexed electrochemical immunosensor developed for the quantification and simultaneous detection of GAA, GBA and GALC. Carbon microarray disposable chips electrodeposited with AuNPs were utilized due to their high conductivity and high surface area that allows the immobilization of more antibodies. Antibodies for GAA, GBA and GALC were immobilized on the sensor for the detection of the proteins. This multiplexed sensor could be utilized in the crucial early diagnosis of LSDs in newborns in order to administer the right treatment.

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