Biotechnology Advances 37 (2019) 634–641 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Biotin interference in immunoassays based on biotin-strept(avidin) chemistry: An emerging threat T ⁎ John H.T. Luonga,b, , Keith B. Malec, Jeremy D. Glennona,b a University College Cork, Irish Separation Science Cluster ISSC Ireland, Innovative Chromatography Group, Western Rd, Cork T12 YN60, Ireland University College Cork, School of Chemistry, College Road, Cork T12 YN60, Ireland c CCA Lab, Hampstead, Quebec H3X 1X5, Canada b A R T I C LE I N FO A B S T R A C T Keywords: Biotin interference Avidin/streptavidin Immunoassays Sandwiched assay Competitive assay Falsely negative Falsely positive Biotin removal Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily doses 100–300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker for cardiac function (β–human chorionic gonadotropin), etc. The biotin level required for serious interference in test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least 1200 ng/mL or 4.9 μM of biotin) in various formats. There is no concrete solution to circumvent the biotin interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in the human body, patients must stop taking biotin supplements for > 48 h before the test. However, this scenario is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples. Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue. 1. Introduction Immunoassays are based on the ability of an antibody to recognize and bind its specific macromolecule in a complex mixture of other molecules. Different assay formats and detection schemes have been developed for a plethora of target analytes of clinical, environmental, and biosecurity importance (Vashist and Luong, 2018). Like many analytical techniques, immunoassays still lack specificity and accuracy, far from perfection. Besides the antibody binding property, the assay specificity is subject to the sample matrix, reagent components, and the assay format. Interfering substances in the specimen alter the measurable analyte level and biotin has become an emerging interferent. Biotin consists of a tetrahydrothiophene ring containing a valeric acid side chain, which is joined to an imidazolidone ring to form eight possible stereoisomers (Scheme 1). Only D(+)-biotin occurs in nature but cannot be synthesized by mammals. As an essential supplement, biotin is part of the B complex group of vitamins that metabolizes amino acids, carbohydrates, and fats. This vitamin biotin might be involved in the regulating transcription or protein expression of different proteins (Pacheco-Alvarez et al., 2002). It is also a critical nutrient for women during pregnancy considering its important role for embryonic growth. Biotin is available from various foods and milk and some intestinal bacteria also synthesize biotin (Combs Jr, 2016). Biotin deficiency results in hair loss (Trüeb, 2008), dry scaly skin, and other symptoms related to fatigue (Osada et al., 2004), insomnia, and depression. Thus, there is increasing marketing of biotin as a remedy for common hair and skin problems, weight loss, glucose metabolism, and boosting energy. High dose biotin therapy has been considered for ⁎ Corresponding author at: University College Cork, Irish Separation Science Cluster ISSC Ireland, Innovative Chromatography Group, Western Rd, Cork T12 YN60, Ireland. E-mail address: j.luong@ucc.ie (J.H.T. Luong). https://doi.org/10.1016/j.biotechadv.2019.03.007 Received 3 January 2019; Received in revised form 21 February 2019; Accepted 8 March 2019 Available online 11 March 2019 0734-9750/ © 2019 Elsevier Inc. All rights reserved. Biotechnology Advances 37 (2019) 634–641 J.H.T. Luong, et al. This review article will address the interference and magnitude of biotin in immunoassays for various biomolecules and biomarkers that play a crucial role in diagnostics. The removal of biotin from the assay sample together with biotin withdrawal might be a useful approach to suppress the interference of biotin and this aspect will also be discussed. In addition, the sensitive and selective detection of biotin in blood samples including using biosensors is discussed. 2. Solid phase immunoassays Scheme 1. Chemical structure of D(+)-biotin, C10H16N2O3S, molar mass of 244.31 g/mol. The human body needs biotin from different food sources to facilitate the conversion of certain nutrients such as fats, carbohydrates, and amino acids into energy. As biotin plays an important role in the health of hair, skin, and nails, it has been formulated in several beauty products. Some bacteria in the intestine of humans also synthesize small amounts of biotin. Biotin forms an irreversible complex with avidin and streptavidin referred to as strept(avidin) and this pair has been extensively used in diversified techniques including commercial immunoassay platforms for clinically important analytes and biomarkers. This unique interaction is also widely used for the preparation of biotinylated antibodies, biotinylated antigens, and strept(avidin)-coated magnetic beads for diversified applications (https://www.thermofisher.com/ca/en/home/ life-science/antibodies/biotin-binding-protein-conjugates.html). The carboxyl group (-COOH) of biotin can be easily bioconjugated with an amino group (-NH2), notably by the popular carbodiimide procedure as shown in Scheme 2 (Hermanson, 2013). Among various carbodiimides, EDC is the most popular choice for conjugating biomolecules containing carboxylates and amines. Water-soluble EDC permits its direct addition to an aqueous solution of biomolecules without prior organic solvent dissolution. The covalent attachment of biotin to proteins, polypeptides, and low molecular weight antigens including thyroid and steroid hormones has minimal effects on the biological and antigenic activities of such biomolecules. This distinct feature enables the use of biotin conjugates as ligands in various immunoassay formats. The solid-phase approach requires only one step and offers high detection sensitivity, specificity, and robustness (Fig. 1). In this simple format, the biotinylated capture antibody is mixed with the specimen and the labeled antibody (luminescent or fluorescent compound, enzyme, isotope), also known as the detection antibody. The “sandwich” format is used to measure large molecules, such as thyroid stimulating hormone, insulin, thyroglobulin, C-peptide, etc. The target is “sandwiched” between the two different antibodies. Consequently, the signal response increases with increasing analyte concentration. Obviously, this “one-step” detection scheme is susceptible to free biotin in the sample since it will compete with biotinylated molecules for streptavidin binding, resulting in falsely low results (Fig. 1A). Based on a very high binding capacity of strept(avidin) coated microparticles, the assay format could provide some binding capacity for free biotin in the sample. The biotin level of healthy subjects is sufficiently low and exhibits negligible interference. However, serious interference becomes pronounced when the biotin level exceeds the threshold concentration for each specific assay. For small analytes, a competitive assay is a better format that involves a capture antibody and a labeled analyte. In this case, both the analyte and the labeled analyte will compete for the binding site of the capture antibody. If the capture antibody is conjugated with biotin, it will bind strept(avidin) coated microparticles, like the sandwich format. For samples with free or low biotin, a high target concentration in the sample will lower the signal-labeled analyte bound to the capture antibody, i.e., the calibration curve is a decreasing curve. Free biotin in samples with high concentrations will compete favorably with the biotinylated capture antibody for the binding site of strept(avidin), resulting in very negligible response signals, known as falsely positive (Fig. 1B). A small population with multiple sclerosis and biotin-related diseases are subject to high biotin dose therapy. The monitoring of biotin in these samples can be accommodated by other analytical procedures including high-performance liquid chromatography equipped with mass spectrometry. various medical problems such as mitochondrial energy metabolism (Depeint et al., 2006), progressive multiple sclerosis (Sedel et al., 2015), and muscle cramps in hemodialysis (Oguma et al., 2012) Despite scientifically unproven benefits, biotin at high doses (> 1 mg) has been used with the expectation to grow hair and nail as mentioned previously. Biotin intake of 1.2 mg shows an average plasma concentration of 15 nM (three hours after an acute dose) and 22 nM after two weeks of prolonged intake. By extrapolation, uptakes of 5–10 mg biotin should result in peak biotin concentrations of 62 nM to 0.124 μM. Even at these levels, most of the immunoassay platforms are still resistant to biotin interference except for troponin T (a biomarker of heart attack) and anti-thyroid antibodies (thyroid disease). The peak serum biotin levels after single doses of 100 mg are 2.024 ± 0.658 μM (Samarasinghe et al., 2017). Thus, overconsumption of biotin (500 mg/ day) results in elevated biotin in blood, micromolar levels, which causes significant interference in immunoassays using the strept(avidin)-biotin interaction. The misdiagnosis and mismanagement of patients were reported for the serious problem of biotin interference in thyroidfunction tests (Trambas et al., 2016). Consequently, biotin becomes an emerging issue in clinical and hospital settings as the immunoassay platform based on biotin-strept(avidin) accounts for 50% of the total immunoassays (221 over 374 methods used by the eight most popular immunoassay platforms in the United States) (Holmes et al., 2017). Unless this problem has been addressed and overcome, immunoassays using the strep(avidin)-biotin chemistry for analyses of target biomarkers and analytes in samples with high biotin are expected to produce aberrant test results. As an example, a recent study illustrates the interference of biotin with a concentration range from 31.35 to 1000 ng/mL or 0.13–4.09 μM (Li et al., 2018) in various immunoassays based on sandwich and competitive formats. As shown in Tables 1-2, the biotin level with serious interference in test results varies significantly from test to test and cannot easily be predicted. Table 1 The concentration of biotin at which interference might be problematic in various assays (Li et al., 2018). Target analytes Serum insulin C-peptide CA (cancer antigen) LH (Luteinizing hormone) Vitamin B12 Vitamin D Prolactin, Folate, CA19-9, CA15-3, CEA (carcinoembryonic antigen), and AFP (alphafetoprotein) Increased (%) 14.48 16.84 Nil Decreased (%) Biotin interference level ng/ml 14.07 14.26 11.13 16.84 750 1000 750 750 750 250 1000 Nil 635 Biotechnology Advances 37 (2019) 634–641 J.H.T. Luong, et al. Table 2 The potential effect of biotin on immunoassays https://www.healthcare.uiowa.edu/path_handbook/Appendix/Chem/BiotinImmunoassayTables.pdf Thyroid markers Thyroid Stimulating Hormone, Reflexive, Thyroid Stimulating Hormone Free T4, Free T3, Total T4, Total T3, Thyroid Peroxidase Antibody, Thyroglobulin Antibodies Hormones Follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, prolactin, growth hormone, insulin, C-peptide Cortisol, estradiol, testosterone, progesterone, dehydroepiandrosterone sulfate Tumor markers Alpha fetoprotein, cancer antigen, carcinoembryonic antigen, carbohydrate antigen, prostate specific antigen, total prostate specific antigen, screening, prostate specific antigen, free, HCG – tumor marker or pregnancy Cardiac markers Troponin T, NT-proBNP Nutritional markers Ferritin Vitamin D, 25-hydroxy, vitamin b12, vitamin b12, reflexive, folate Infectious disease serologies HIV antigen/antibody combo, hepatitis c virus antibody, hepatitis a antibody, total, hepatitis a antibody, IgM, hepatitis b surface antigen, hepatitis b surface antibody, hepatitis b antigen, hepatitis b core antibody, IgM. Hepatitis B core antibody, total (LAB622) Pregnancy-related markers Pregnancy screen, qualitative, HCG – pregnancy, HCG – tumor marker or pregnancy Therapeutic Drugs Digoxin Other proteins Immunoglobulin E, myoglobin, sex hormone binding globulin R O Falsely decreases Falsely increases Falsely decreases Falsely decreases Falsely decreases Falsely increases Falsely decreases Falsely increases Falsely decreases Falsely increases Falsely decreases O R N=C=N NH2 R1 Falsely decreases Falsely increases N R1 OH H Biomolecule CH3 Cl NH N=C=N H H3C CH3 EDC = 1-Ethyl-3-3(dimethylaminopropyl) Carbodiimide Hydrochloride, MW 191.7 Scheme 2. Covalent attachment of the carboxyl group to the amino group by the popular carbodiimide coupling procedure. 3. Avidin and its analogs isolated from Streptomyces avidinii is smaller (53 kDa) compared to avidin and lacks the glycoprotein portion found on avidin. Captavidin has a nitrated tyrosine in its biotin-binding site (Takakura et al., 2009), resulting in its significantly lower affinity to biotin, compared with other avidin analogs (Table 3). The captavidin-biotin pair can be dissociated at pH 10 instead of the necessity of 8 M guanidine hydrochloride, pH 1.5 for the avidin-biotin complex. A recombinant avidin, known as tamavidin, is derived from E. coli-expressed mushroom (Pleurotus cornucopiae) with much lower affinity to biotin, compared to Tetrameric avidin (MW = 67–68 kDa), commonly found in egg whites, is synthesized in the oviducts of various animals, e.g., birds, reptiles, and amphibians whereas dimeric avidin is also found in some bacteria, e.g., Rhizavidin from Rhizobium etli (Helppolainen et al., 2007). Neutravidin or deglycosylated avidin retains its affinity to biotin with minimal nonspecific binding properties, particularly, the nonbinding of sugar binding proteins. Like neutravidin, streptavidin Fig. 1. (A) (Upper) Microparticles are coated with strept (avidin). The capture antibody for a target analyte is bioconjugated with biotin. The specific binding between biotin-strept(avidin) leads to the formation of a tertiary complex. The capture antibody then binds the target compound. (Lower) Free biotin in the sample will compete with the biotinylated capture antibody for binding to strept (avidin) coated on microparticles, leading to erroneous results. This format is known as “sandwich” assays for large molecules. (B). The target analyte is labeled with a detectable signal and this format is applicable for detecting small molecules. Without biotin in the sample, a high target concentration in the sample will lower the signal-labeled analyte bound to the capture antibody. For samples with high biotin levels, free biotin will compete favorably with the biotinylated capture antibody for the binding site of strept(avidin), resulting in very negligible response signals, known as falsely positive. 636 Biotechnology Advances 37 (2019) 634–641 J.H.T. Luong, et al. Table 3 Some key properties of avidin and its analogs. Properties Avidin Neutravidin Streptavidin Captavidin Tamavidin MW (kDa) Affinity for biotin (Kd,M−1) Binding sites for biotin Isoelectric point (pI) 67–68 10−15 4 10 60 10−15 4 6.3 53 10−15–10−14 4 6.8–7.5 67–68 10−9 4 60 2.8–4.4 × 10−7 (Ref. 4) 4 crosslinker” since EDC does not become part of the final amide-bond crosslink between the two molecules (Hermanson, 2013). The attachment of biotin to various molecules is termed as “biotinylation”, which plays an important role in other analytical techniques to probe protein localization-interaction, protein, DNA transcription, replication, etc. The BCAb forms a stable complex with streptavidin and offers accessible binding sites for both the analyte and the conjugated analyte. The activity of the conjugated analyte is measured and related to the analyte concentration. The response signal is high for low analyte concentration and vice versa as the response signal is inversely proportional to the analyte concentration. In the presence of elevated biotin, these free molecules bind streptavidin strongly and pull it out of the beads. The biotinylated Ab still binds the analyte and the conjugated analyte, but all materials will be washed out during the washing step. The response signal becomes very small or negligible, reflecting a high level of the analyte in the calibration curve, falsely positive. This format is used to target small molecules such as testosterone, estradiol, cortisol, steroids, T3 (triiodothyronine) and T4 (thyroxine)-hormones produced by the thyroid gland, hydroxyvitamin D, etc. In sandwich assays, the Ab for a target analyte is biotinylated, designated as the capture or primary Ab. The Ab is also conjugated with an enzyme, designated as detection or secondary antibody. With biotinfree samples, the biotinylated capture Ab binds streptavidin and the antigen at two different binding sites. The labeled Ab then forms a tertiary complex with the antigen and the capture Ab. The activity of the labeled enzyme is quantified and related to the amount of the antigen. Based on a double epitopic recognition, sandwich assays are also known as “two-site immunoassays”. In the presence of free biotin in a sample, this molecule competes with the biotinylated capture antibody for binding streptavidin. As a result, the density of the tertiary complex is greatly reduced, resulting in an underestimation of the antigen in the sample or false negative (Fig. 1A). In clinical settings, the sandwich assay is applied to measure fairly large molecules to very large molecules with molecular weights ranging from 3 kDa to 650 kDa: stimulating hormone (TSH, thyrotropin: MW = 28,000), pituitary glycoprotein hormones (PGH), human chorionic gonadotropin (HCG: MW = 36,700), parathyroid hormone (PTH, MW = 9500), insulin-like growth factor-1 (IGF1, MW = 7649), insulin (MW = 5808), thyroglobulin (MW = 650,000), and C-peptide (MW = 3020). There are three human pituitary glycoprotein hormones: luteinizing hormone (LH, MW = 30,00 for animals), follicle-stimulating hormone (FSH, MW = 35,500), and thyrotropin (TS, MW ~ 28,000). A guidance for tests of serious diseases such as HIV and hepatitis C virus needs to be developed to provide proper results. This concern is also extended to the assays of cardiac biomarkers and the assessment of tumor recurrence. Slight skewing of the results might lead to erroneous treatment and devastating emotions. its analogs (Takakura et al., 2010; Takakura et al., 2013). Of importance is the stability of the biotin-strept(avidin) complex under extreme pH and temperature and its resistant to various organic solvents and denaturing agents. Detailed dimensional (3D) structures of both avidin and streptavidin in complex with D-biotin can be found in the literature (Livnah et al., 1993a, 1993b). Both tetrameric avidin and streptavidin have four identical subunits, with each subunit containing a single biotin-binding site. However, avidin is glycosylated with one disulfide bridge and two methionine residues whereas streptavidin is a non-glycoprotein without sulfur-containing residues. The amino acid compositions of these two proteins are also very different. Avidin has a single tyrosine (Tyr) residue whereas streptavidin has six Tyr per subunit. The avidin (Tyr-33) is in the primary sequence (30Thr-Gly-ThrTyr-Ile-Thr-Ala-Val) and located in a position similar to one of the Tyr residues (Tyr-43) of streptavidin (40Thr-Gly-Thr-Tyr-Glu-Ser-Ala-Val). Upon the binding of biotin, strept(avidin) changes its conformation, stability, and flexibility (Livnah et al., 1993a, 1993b; Weber et al., 1992; Gonzalez et al., 1999). Biotin interacts strongly with avidin in a loop located between β-strands 3–4, its most flexible part. Of importance is the role of the single tyrosine residue (Tyr-33) in the avidin subunit, which is attributed to the biotin-binding site. Modification of avidin by p-nitrobenzenesulfonyl fluoride, a tyrosine-specific reagent (Liao et al., 1982) results in the complete loss of its biotin-binding (0.5 mol of tyrosine residue/mol of avidin subunit) (Tsou, 1962). The tyrosine residues may also stabilize the native protein structure. For streptavidin, the modification of 3 mol of tyrosine residue/mol of subunit is required to nullify its biotin-binding activity (Livnah et al., 1993a, 1993b). Apparently, Tyr-43 (major fraction) and Tyr-54 (minor fraction) are also involved in the binding of biotin. Streptavidin is preferred over avidin in immunoassays due to its lower non-specific binding properties (Chivers et al., 2010) and comparable affinity to biotinylated molecules. Captavidin and tamavidin with much lower affinity to biotin offer the possibility to dissociate the complex when formed. They might be more receptive in biosensing schemes, bioseparation or other applications that require the dissociation of the avidin-biotin pair for repeated uses. Commercial avidin, streptavidin, and neutravidin with high purity bind 12 μg (0.05 μmol) of biotin (MW = 244) per mg protein or 1.4 × 10−2 μmol (MW = 50 kDa–70 kDa), corresponding to a biotin to avidin binding ratio of 3.6, approaching the maximum value of 4 (Table 3). 4. Immunoassays using the biotin/streptavidin interaction Streptavidin is preferred over avidin to minimize nonspecific protein binding despite its higher cost and there are several formats based on the streptavidin-biotin chemistry in immunoassays. In competitive assays, streptavidin-coated beads, a conjugated analyte (normally with an enzyme, e.g., horseradish peroxidase), and a biotinylated capture antibody (BCAb) are prepared. Compared to avidin, biotin is a significantly smaller molecule (MW = 244) with a carboxylic group. The presence of this group has been exploited to conjugate with a plethora of proteins, enzymes, and other molecules for diversified applications. Of interest is the use of EDC (1-ethyl-3-(−3-dimethylaminopropyl) carbodiimide hydrochloride) and other carbodiimides to effect direct conjugation of the biotin carboxyl to the amino group of a second biomolecule (protein, enzyme, etc.). This is called a “zero length 5. Issues of overconsumption and high biotin dose therapy Biotin (vitamin H), literally stems from German “Haut und Haar”, i.e., “skin and hair”. To date, over-consumption of biotin is inspired by lofty claims that biotin helps grow healthy and strong hair, skin and nails. Supporting scientific evidence for this claim is very limited, however, biotin improves the keratin infrastructure, a basic protein of 637 Biotechnology Advances 37 (2019) 634–641 J.H.T. Luong, et al. Keenan, 2013). The risk of side effects can be reduced by taking biotinsupplements with foods. Its negative side effects are also rare since it is easily excreted in urine and feces. In contrast, biotin might improve cognitive function, reduce inflammation, lower blood sugar in diabetes, decrease “bad” LDL cholesterol and increase “good” HDL cholesterol (https://www.healthline.com/health/biotin-hair-growth#otherbenefits). Nevertheless, significant scientific proofs are needed to support or refute the side-effects of this water-soluble vitamin as beauty supplements in high dosage. Table 4 Biotin interference and its threshold concentration reported for seven commercial systems. Commercial automated system Total test Total vulnerable Biotin concentration (nM) Roche Elecsys® Ortho Vitros® Siemens Dimension® Siemens Centaur® Beckman Coulter Access®/ DXI® Abbott Architect i2000®a Diasorin Liaison XL®b 81 43 26 67 48 81 29 21 18 14 29–491 10–82 205–8200 41–4090 41–1000 46 42 2 0 120 Not available 6. Interference of biotin and the removal of biotin Biotin ranging from 0.12 to 0.36 nM (Zempleni et al., 2009) in blood of healthy subjects falls into the tolerant level of the assay format and has a negligible interfering effect on the avidin/streptavidin-biotin technology (Kwok et al., 2012). Elevated biotin in blood had been limited to a small population until biotin megadoses became commonplace for non-medical purposes. Enhanced biotin levels in plasma have been observed for a daily consumption of 300 μg of biotin for 1 week and then 900 μg for another week (Bitsch et al., 1989). Over 1 mg/day of biotin consumption often causes falsely-low or falsely-high test results, depending on the assay format. The magnitude of interference and the threshold of biotin concentration, however, are also variable and dependent upon the type of assay and the target molecules. For instance, the assay of anti-TPO (thyroid peroxidase) is vulnerable to plasma biotin above 40.9 nM, compared to 491 nM for carcinoembryonic antigen (CEA) (https://www.exeterlaboratory.com/ biotin-alert-potential-assay-interference). Table 4 illustrates the results of biotin interference in all immunoassays performed by seven commercial immunoassay systems. In general, the test is vulnerable to biotin presence when the procedure is based on having a streptavidin/ biotin reaction, an anti-biotin/biotin reaction, or a pre-bound avidin/ streptavidin or biotin/anti-biotin reagent as part of the analysis. In general, the interference is considered when the spiked biotin in a sample causes ≥ ± 10% variation of the expected result. The two systems (Abbott and Diasorin) are not based on strept(avidin)-biotin technology or involve any chemistry related to biotin. At first glance, biotin from samples can be removed by avidin/ streptavidin immobilized on insoluble matrices such as magnetic particles, polymers (e.g. agarose beads), silica, etc. Such matrices, commercially available from different suppliers, are added to the microplate together with the sample. This step is time-consuming, about 1 h for incubation and the removal of the biotin-avidin complex. Strept (avidin) bound polymers could be packed in a small syringe to facilitate the loading of a biotin-containing sample. Of course, such modifications will require extensive revalidation which is time-consuming and expensive. The “full-proof” biotin-removal capacity of streptavidinagarose beads is still a subject of future endeavors. Nevertheless, clinical and hospital settings must be aware of elevated biotin levels in the assay samples to prevent any misdiagnosis and/or inappropriate therapy. Other solutions include the dilution of the sample with a validated assay diluent or using a different platform known to be unaffected by biotin interference. Biotin withdrawal is the easiest option to reduce the financial burdens associated with high-dose biotin on the health care system. With a half-life of ~ 2 h in low dose consumption, most of biotin should be cleared from the body within 4–5 h. However, a period of two to five days might be required for patients with high biotin dose. It should be noted that this period can be over 15 days, e.g., thyroid function tests (TFTs) (Koehler et al., 2018) or months in other cases. This strategy is not an option for patients with MS or other biotin related diseases. Of course, clinical or hospital settings are equipped with radio immunoassay (RIA) and gas chromatography–mass spectrometry/liquid chromatography-mass spectrometry to handle such analyses. Lastly, the replacement of biotin-strept(avidin) by a new pair with the same binding constant opens a new approach in immunoassays. a NB The Abbott Architect i2000® is not based on strept(avidin)-biotin chemistry. b As an example, the method for hCG is a sandwich chemiluminescence immunoassay. A specific mouse monoclonal antibody is coated on the magnetic particles (solid phase), another monoclonal antibody is linked to an isoluminol derivative (isoluminol-antibody conjugate (https://www.accessdata.fda.gov/ cdrh_docs/pdf13/K131037.pdf). hair, skin, and nails. As a result, women and “bald” men have become obsessive toward biotin consumption as high as 1250–2500 μg twice daily to beautify their skin, nail, and hair. The recommended dose of biotin for adults by the Institute of Medicine (US) is only 30 μg per day (Ross et al., 2016). Another subject of debate is the efficacy of high-dose biotin therapy for patients with relapsing and progressive multiple sclerosis (MS). Myelin is impaired in MS patients, a “lipid-rich” compound that protects the nerve cells. Dimethyl fumarate (DMF), used for the treatment of psoriasis, is approved to treat patients with relapsing-remitting multiple sclerosis (MS) (Bomprezzi, 2015). DMF exposure offers the potential cytoprotection of neurons, oligodendrocytes, and glial cells. Another FDA-approved immunosuppressive drug for progressive MS in 2017 is Ocrelizumab (trade name Ocrevus). This humanized anti-CD20 monoclonal antibody targets the CD20 marker on B lymphocytes (McGinley et al., 2017). Clinical trials are still in progress to establish its efficacy, safety cancer risks and any adverse effects on pregnant women and children they might bear. This drug is exorbitant, priced at $65,000 (annual cost, for two infusions per year (Ron Winslow, March 28, 2017). “After 40-year odyssey, first drug for aggressive MS wins FDA approval”), thus, the search is on for more effective treatments and affordable drugs. Biotin is very inexpensive and has not been known for any reported serious side effects or cytotoxicity. Biotin promotes the production of myelin and provides energy for neurons. Many people with multiple sclerosis (MS) use vitamins, particularly B vitamins to manage their symptoms to convert food into energy for supporting the nervous system. Biotin (B7 or vitamin H), is one of the B complex vitamins and is essential for human health. Biotin activates several key enzymes and helps the body to produce more myelin for cell-cell communication, reducing the level of disability. MS patients often take 300 mg per day. Biotin therapy is extended to people with biotinidase deficiency and this disease often commences within months of birth. People with this disorder lack biotinidase, an enzyme that recycles biotin several times before its discharge as waste. Other rare genetic diseases that cause biotin deficiency include holocarboxylase synthetase deficiency, biotin transport deficiency, and the more common phenylketonuria. The daily dose of such patients is 10–40 mg (Henry et al., 1996; Wijeratne et al., 2012). The plausible side effect of biotin deserves a brief comment here considering the very high levels of biotin in some beauty products. In most cases, no serious adverse effects have been reported but minor side effects include nausea, cramping, and diarrhea. Biotin consumption in high doses might affect the reproductive female performance by interfering with the synthesis of estrogen and progesterone (Wallig and 638 Biotechnology Advances 37 (2019) 634–641 J.H.T. Luong, et al. et al., 1991), also resulting in an absorption peak around 500 nm. Excess albumin in the sample might interfere with the assay. In blood plasma, 81% is free, 12% is covalently bound, and 7% is reversibly bound (Mock and Malik, 1992). Thus, the measurement of biotin in blood plasma without sample treatment will represent 80% of the total biotin. Of notice are two major biotin metabolites: biotin sulfoxide (BSO) and bisnorbiotin (BNB). In the cerebrospinal fluid of children, biotin accounts for 42 ± 16%, BSO for 41 ± 12%, and BNB for 8 ± 14% of the total biotin (Bogusiewicz et al., 2008). High-performance liquid chromatography (HPLC) on a bonded phase C18 column can be used to resolve biotin and its analogs. Biotin exhibits no absorption or fluorescence properties, so such compounds must be derivatized to ω,4-dibromoacetophenone esters for UV detection or 4-bromomethylmethoxy-coumarin derivatives for fluorometric detection (excitation/emission wavelengths at 360 nm and 410 nm) (https://www.thermofisher.com/ca/en/home/life-science/cell analysis/fluorophores/coumarin.html.). HPLC (C18 column) with postcolumn derivatization, using o-phthalaldehyde and 3-mercaptopropionic acid (3-MPA) analyzes for biotin in pharmaceutical preparations with a detection limit of 10 ng per injection (Nojiri et al., 1998). Direct determination of biotin in multivitamin pharmaceutical preparations is feasible by HPLC using electrochemical detection (the type of electrode is not specified) (Kamata et al., 1986). The ultimate system in clinical and hospital settings is HPLC-MS/MS. Some pre-treatments are required and electrospray with positive ionization has been proven as the most sensitive ionization method. There are two prominent SRM (specialized pro-resolving mediators) transitions observed for biotin, m/z 245 → 227, and 245 → 114 but only the first one is sufficiently intense for quantification (Holler et al., 2006). HPLC using reversed-phase and anion-exchange chromatographic conditions can also resolve biotin and its analogs. Anion-exchange separations give generally shorter retention times and greater resolution between biotin I- and d-sulfoxide, compared to reversed-phase separations. Such lab-based methods are time-consuming and expensive but provide accurate levels of the target analytes in the plasma of MS patients. This disease is no longer rare with the global median prevalence of 33/100,000 and could be higher in specific parts of the world (Thompson et al., 2014). Electroanalysis with low cost, ease of fabrication/miniaturization, and high detection sensitivity might be useful for the detection of biotin in blood. However, the progress in this area is very limited. There are only a few literature reports related to electroanalysis/electrocatalysis of biotin (Lauw et al., 2013; Marin et al., 1977; Serna et al., 1973). Recently, a simple Nafion modified boron-doped diamond (BDD) electrode detects biotin as low as 5–10 nM (Buzid et al., 2018). However, it lacks detection selectivity as other endogenous species in biological samples, e.g., amino acids, vitamins, dopamine, etc. are also oxidized in this detection scheme. Thus, the issue of detection selectivity is still very challenging considering the presence of endogenous electroactive species such as vitamins, drugs, and their metabolites, amino acids, etc. in blood samples. Of notice is the use of captavidin as a regenerable biorecognition element on boron-doped diamond for biotin sensing (Buzid et al., 2019). The biotin-avidin binding occurs via hydrogen bonding between the carbonyl group on the ureido ring of the former and the single tyrosine (Tyr-33, pKa = 10.5) of the latter at pH below this pKa (Morag et al., 1996). For captavidin, three of the four tyrosine moieties are nitrated to nitroso-avidin with a pKa value of ~7.2. Thus, its binding to biotin is weaker compared to avidin at pH below 10. As shown in Table 3, the association constant of the captavidin-biotin pair is only 10−9 M compared to 10−15 M (González et al., 1997) of the strept (avidin)-biotin counterpart. Above pH 10, the hydroxyl groups of the tyrosine moieties ionize, affecting the hydrogen disruption to release biotin. This behavior has been exploited for the fabrication of a regenerable biosensor for the detection of biotin in blood plasma with a detection limit of below 1 nM. In this impedance sensing scheme, captavidin irreversibly adsorbs on carboxymethyl cellulose to form a Cucurbiturils represent emerging molecules (Mock, 1995) that can bind several molecules, e.g., diamantane diammonium ion, with Ka = 7.2 × 10−17 M−1 in D2O. Cucurbiturils with different sizes can be synthesized from the acid-catalyzed condensation of glycoluril and formaldehyde. Albeit the chemistry of cucurbiturils have received considerable attention (Shetty et al., 2015; Hwang et al., 2007), the implementation of these materials and their counterparts is a long-term endeavor. First, it remains to investigate their non-specific protein binding and plausible interferences from biotin, vitamins, and other endogenous molecules in clinical and biological samples. The use of magnetic particles (micro or nanoscale), consisting of an inorganic core of iron oxide (magnetite (Fe3O4), maghemite (Fe2O3) or other insoluble ferrites), deserves a brief comment here due to their commercial availability and facile bioconjugation. Coating magnetic particles with a polymer, organic molecule or biomolecule imparts functional groups such as amino and carboxylic acids to facilitate subsequent conjugations. Such materials conjugated with strept(avidin) are also attractive for their use in the removal of biotin in the sample. Nanoparticles of iron oxides (5–15 nm in diameter) are superparamagnetic whereas microparticles are ferromagnetic. Coating magnetic particles with a polymer alters their size and magnetic properties. For applications in the separation technique or biotin removal, small magnetic monodomain nanoparticles are preferred because they do not possess remanence (remanent magnetization or residual magnetism) when the magnetic field is removed (Leslie-Pelecky and Rieke, 1996). Nanoparticles of metal oxides, e.g., magnetite Fe3O4 and maghemite γFe2O3 are simply prepared by the alkaline coprecipitation of ferric and ferrous salts (Massart and Cabuil, 1987). Magnetic polymer beads with various functional groups can be prepared by coprecipitation of iron salts directly in a pertinent polymer matrix. In general, the size of magnetic particles becomes smaller than in the absence of polymer (Pardoe et al., 2001). In principle, immunoassays using these “pre-bound” reagents followed by sample addition are expected to be resistant to, or insignificantly affected by biotin interference. However, thorough investigation and validation are still needed to ascertain that these expectations are supported by the results of interference studies. Immunoassay manufacturers are expected to conduct this protocol for various target analytes to establish the tolerable biotin concentration for each analyte. Similarly, the pre-removal of biotin in the sample using strep(avidin) coated polymers or magnetic particles is still subject to intensive investigation to iron out any plausible interference due to non-specific interaction/adsorption of the target analyte or other endogenous species with strept(avidin) coated carriers. 7. Measurement of biotin Rapid screening of biotin is desirable before the sample is subject to immunoassays to provide a warning to plausibly falsely negative or falsely positive outcomes. Such information may be used for more accurate risk assessment in predicting the effects of biotin. The HABA (4′hydroxyazobenzene-2-carboxylic acid) method provides a colorimetric method to estimate the biotin concentration in a solution. HABA has an absorption peak at 348 nm and binds avidin relatively weakly (Kd = 5.8 × 10−6 M) (Green, 1970) to form an emerging peak at 500 nm. HABA binds to avidin as a hydrazone tautomer, involving an intramolecular hydrogen bond and the loss of planarity (Livnah et al., 1993a, 1993b). Similarly, streptavidin binds HABA as the hydrazone tautomer with a lower Kd (10−4 M (Green, 1970). Some HABA derivatives bind avidin more strongly but their absorption (extinction) coefficients are low (Repo et al., 2006). Biotin can easily dissociate HABA from the HABA-avidin complex due to its significantly higher affinity for avidin (Kd = 10−15 M). The decrease of the absorption peak at 500 nm is followed and related to the biotin concentration. This assay has a linear range from 2 to 16 μM. Of importance is the non-specific binding of avidin with other proteins, e.g., serum albumin (Tarnoky 639 Biotechnology Advances 37 (2019) 634–641 J.H.T. Luong, et al. presence of elevated biotin in bloods affects immunoassays using the biotin-strept(avidin) pair for heart failure, pregnancy, cancer, and irondeficiency anemia. This issue is “somewhat” complicated as the level of biotin interference is dependent on the tested analyte. As a few examples (Schauss, 2018), the hCG (human chorionic gonadotropin), PTH (parathyroid hormone), and NTproBNP (N-terminal pro–B-type natriuretic peptide) assays are significantly interfered by > 5 ng/mL (20 nM) of biotin concentration whereas the Troponin-T assay experiences no interference from biotin up to 100 ng/mL (0.4 μM). Biotin is an emerging interferent but manufacturers for immunoassay equipment and chemicals are still looking for some concrete solutions. It will take time and money to find a bold solution and clinical validation. At best, the subject must abstain from biotin intake for 2 days or longer before the test, whereas clinical laboratories try their best by running the same sample on different immunoassay platforms to validate the results. Sample dilution can alleviate this problem but not beyond the detection sensitivity for the target analyte. Patients should declare whether they have consumed biotin-containing supplements prior to having sample collection, considering a quick and inexpensive method for analysis of plasma biotin is still not available. Biotin metabolites may interfere in some assays and this task deserves further studies. This issue of biotin interference is particularly critical for patients in emergency situations considering a quick test for biotin is still not available to confirm whether the patient has been subjected to high-dose biotin. Apparently, an extensive communication campaign to educate physicians, laboratorians, and patients must be fostered to publicize the emerging issue of elevated biotin interference in immunoassays. Supraphysiologic biotin intake will not be diminished any time soon, particularly for MS patients and for patients with other inherited metabolic diseases, who are subject to high dose biotin therapy. Furthermore, over-consumption of biotin also stems from claims of its benefit for healthy hair and nail growth. Thus, elevated biotin in blood remains problematical for immunoassays using the avidin/streptavidinbiotin chemistry. regenerable biorecognition element for biotin. This layer is then retained and stabilized on a boron-doped diamond electrode by a Nafion film. The biosensor has two distinct features: captavidin confers detection specificity and regenerability whereas the negatively charged Nafion and carboxymethylcellulose layers circumvent the diffusion of endogenous electroactive species. Indeed, the literature also covers the use of captavidin in surface plasmon resonance, SPR (Garcia-Aljaro et al., 2009) and carbon nanotube (SWNT) field-effect transistors, FETs (Munzer et al., 2014). With SPR, biotinylated antibodies can be successfully subject to up to nine serial capture-release events from the captavidin-functionalized surface, i.e., captividin is not exploited for the binding and detection of biotin. For the work related to FET, the reversible captavidin binding with pyrene-biotin functionalized SWNT FETs has been demonstrated. This biosensing scheme offers to probe the dissociation constant of captavidin and differentiate between two different biotin-binding molecules, streptavidin and Neutravidin based on the pH-dependent sensor response. Gold nanoparticle decorated SWNT FETs are functionalized with biotin to display reversible captavidin binding. This sensing scheme could be a promising platform for the detection of proteins based on enhanced Raman spectroscopy (Munzer et al., 2014). Gold nanoparticles (AuNPs) have been used together with magnetic beads in a competitive immunoassay format for the detection of biotin (Lin et al., 2019). The assay is based on anti-biotin antibody-modified magnetic beads (Ab-MBs) and biotinylated thiol-DNA AuNPs (biotinAuNPs). Without biotin in the sample, biotin-AuNPs bound to Ab-MBs are retained by an external magnetic field and the solution is transparent. For the assay sample with biotin, free biotin will compete with biotin-AuNPs and bind the Ab-MBs and the complex is retained by the magnetic field. Unbound biotin-AuNPs are still floating in the solution, resulting in a red color or surface plasmon resonance at 530 nm. The detection limit of this method for biotin is only 2 pmol/100 μL of the total sample, i.e., 20 nM. Several proteins, amino acids, and biomolecules will bind AuNPs to cause significant interferences (Zhong et al., 2004). Albeit this method has been tested for some food samples, its applicability for the analysis of biotin in blood or blood plasma remains to be confirmed. The colorimetric assay strategy for avidin and biotin interactions can be probed by a colorimetric assay using AuNPs (Shi et al., 2018). In brief, biotin-ssDNA specifically bound to avidin with strong affinity is protected from hydrolysis by exonuclease I (Exo I). The biotin-ssDNA (negative charge) would attach to the surface of AuNPs (positive charge) in high salt solution through electrostatic interactions to prevent the aggregation of AuNPs. The surface plasmon resonance of AuNPs at 520 nm increases gradually with a red color with increasing avidin in the assay sample. This method aims for the detection of avidin with a detection limit of 4 × 10−3 μg/mL. Therefore, considerable efforts are still needed to use this concept for the detection of biotin. References Bitsch, R., Salz, I., Hötzel, D., 1989. Studies on bioavailability of oral biotin doses for humans. Int. J. Vitam. Nutr. Res. 59 (1), 65–71. Bogusiewicz, A., Stratton, S.L., Ellison, D.A., Mock, D.M., 2008. Biotin accounts for less than half of all biotin and biotin metabolites in the cerebrospinal fluid of children. Am. J. Clin. Nutr. 88 (5), 1291–1296. Bomprezzi, R., 2015. Dimethyl fumarate in the treatment of relapsing-remitting multiple sclerosis: an overview. Ther. Adv. Neurol. 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