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

Lysosomal Storage Disorders (LSD) are a group of inherited genetic diseases that cause the accumulation of lipids 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 accumulation of substrates leading to cell destruction and dysfunction, in turn, causing tissue dysfunction2.6. The severity of LSD depends on the nature and amount of accumulated substrate6. LSD patients appear normal at birth, but develop symptoms early in childhoodseven. Neurological symptoms include brainstem dysfunction and seizures, and peripheral symptoms include kidney and heart damage, muscle atrophy, ophthalmic disease, enlarged liver and spleen, and irregular bone development.8. However, treatment is available for most LSD if discovered early in childhood, such as enzyme replacement therapy (ERT), bone marrow or stem cell transplantation, and gene therapy. Therefore, early detection of LSD is crucial.6.7.

Pompe disease (type II glycogen storage disease) is an SIDS caused by a deficiency in the lysosomal enzyme acid α-glucosidase (GAA). GAA is needed to break down glycogen into glucose, so its deficiency leads to accumulation of glycogen in organelles9.10. As a pump treatment, acid α-glucosidase enzyme is given to patients as ERT, it breaks down glycogen into glucose to reduce its accumulation in cells, however, treatment with acid α-glucosidase should be started as early as possible in infants11. Another LSD is Krabbe’s disease (KD) or globoid cell leukodystrophy (GCL), is a neurological condition caused by galactocerebrosidase (GALC) deficiency. The 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 untreated12.13. Gaucher disease is the most common sphingolipidosis, it results from β-glucocerebrosidase (GBA) deficiency14it causes the accumulation of glucosylceramide in macrophages15and it is classified into three main subtypes based on the presence or absence of neurological involvement16. Of all LSDs, Pompe’s, Gaucher’s and Krabbe’s diseases are the most common and have severe symptoms that end in death. However, these diseases have treatments available and are detectable in their childhood stage. Therefore, if detected and diagnosed early in the patient’s infantile phase, treatments can be administered accordingly.

It has been proven that the initial identification of patients with LSD can be achieved by immunoquantification of enzymes and lysosomal proteins, since mutations lead not only to a deficiency in enzyme activity, but also to a decrease in the amount of protein.seven. Deficient patients have protein levels below the threshold concentration which is approximately 3 to 100 ng/ml in healthy individuals17. Methods previously used for the quantification of some LSD-related proteins have been reported, such as fluorescence, tandem mass spectrometry (LC-MS/MS) and immunoassays like enzyme immunoassay (ELISA).2.7. Nevertheless, these methods are known to be time-consuming and time-consuming to analyze, expensive, require specialist laboratories, and require a large volume of samples, making them not ideal for point-of-care (POCT) testing.18. POCT allows rapid access to results thus providing faster monitoring, choice of treatment, prognosis and diagnosis of diseases, resulting in better decision making which is often vital to patient health. To perform POCT in the most efficient way, more cost-effective and faster methods are being developed.19.

To speed up and facilitate clinical diagnosis and POCT, multiple analysis of different biomarkers yields faster and more accurate results. Thus, multiplexed analysis using a single analytical device holds great promise in upgrading and simplifying diagnostic procedures, as it provides more data, faster. Multiplexed detection offers many advantages such as using less sample volume, less average analysis time, more statistically reliable conclusions and more informative detection results20.

Biosensors are evolving to become an interesting, cheaper, simpler and more sensitive alternative to conventional detection methods for diagnostic purposes. Specifically, electrochemical immunosensors are the focus of much research in the field of biomedical research and diagnostics, due to their high sensitivity, rapid response, minimization of sample volume used, ability to be miniaturized and their multiplexing capacity. Electrodeposition of electrochemical immunosensors with gold nanoparticles (AuNPs) by chronoamperometry, improves its performance via improved electron transfer rate and catalytic activity of the sensor21. This is done by reducing HAuCl4 using potassium nitrate. AuNPs increase surface area allowing more antibodies to immobilize on the surface of the transducer, resulting in significantly higher signal and sensitivity. Electrodes modified with gold nanoparticles demonstrated a three-fold increase in electroactive area, resulting in increased functional density of biomolecules as well as improved electron exchange and sensitivity22,23,24.

In this work, we report a new multiplexed electrochemical immunosensor developed for the quantification and simultaneous detection of GAA, GBA and GALC. Disposable carbon microarray chips electrodeposited with AuNPs were used due to their high conductivity and large surface area which allows immobilization of more antibodies. Antibodies for GAA, GBA and GALC were immobilized on the sensor for protein detection. This multiplexed sensor could be used in crucial early diagnosis of LSD in newborns to deliver the right treatment.

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