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The method is based on developing the enzymatic hydrolysis and oxidation of cholesterol at microscale size using both enzymes (cholesterol esterase (ChE) and cholesterol oxidase (ChOx)) immobilized on magnetic nanoparticles (MNPs). The biocatalyst reactions were followed by monitoring the fluorescence decreasing by the naphtofluorescein (NF) oxidation in the presence of the previous H<jats:sub>2<\/jats:sub>O<jats:sub>2<\/jats:sub> formed. This microfluidic biosensor supposes the physical integration of a minimal MRE\u00b5R as a bioactive enzyme area and the focused BFOB connected with the spectrofluorometer detector. The MRE\u00b5R was formed by a 1\u00a0mm length of magnetic retained 2:1 ChE-MNP\/ChOx-MNP mixture. The dynamic range of the calibration graph was 0.005\u201310\u00a0mmol\u00a0L<jats:sup>\u22121<\/jats:sup>, expressed as total cholesterol concentration with a detection limit of 1.1\u00a0\u00b5mol L<jats:sup>\u22121<\/jats:sup> (<jats:italic>r<\/jats:italic><jats:sup>2<\/jats:sup>\u2009=\u20090.9999, <jats:italic>s<\/jats:italic><jats:sub><jats:italic>y<\/jats:italic>\/<jats:italic>x<\/jats:italic><\/jats:sub>\u2009=\u20090.03, <jats:italic>n<\/jats:italic>\u2009=\u200910, <jats:italic>r<\/jats:italic>\u2009=\u20093). The precision expressed as the relative standard deviation (RSD%) was between 1.3 and 2.1%. The microfluidic-based biosensors showed a sampling frequency estimated at 30\u00a0h<jats:sup>\u22121<\/jats:sup>. The method was applied to determine cholesterol in serum samples with recovery values between 94.8 and 102%. The results of the cholesterol determination in serum were also tested by correlation with those obtained using the other two previous methods.<\/jats:p>\n                <jats:p><jats:bold>Graphical abstract<\/jats:bold><\/jats:p>","DOI":"10.1007\/s00604-023-05894-w","type":"journal-article","created":{"date-parts":[[2023,7,19]],"date-time":"2023-07-19T03:29:59Z","timestamp":1689737399000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["An integrated microfluidic-based biosensor using a magnetically controlled MNPs-enzyme microreactor to determine cholesterol in serum with fluorometric detection"],"prefix":"10.1007","volume":"190","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1116-0427","authenticated-orcid":false,"given":"Vanesa","family":"Rom\u00e1n-Pizarro","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3240-5769","authenticated-orcid":false,"given":"\u00c1ngela","family":"\u00c9cija-Arenas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8443-1358","authenticated-orcid":false,"given":"Juan M.","family":"Fern\u00e1ndez-Romero","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2023,7,19]]},"reference":[{"key":"5894_CR1","doi-asserted-by":"publisher","first-page":"1515","DOI":"10.4158\/EP-2020-0347","volume":"26","author":"D Schade","year":"2020","unstructured":"Schade D, Shey L, Eaton RF (2020) Cholesterol review: a metabolically important molecule. 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