Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.
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The Python code used to analyse bead images is provided at https://github.com/beirami/rt-elisa.
Hamburg, M. A. & Collins, F. S. The path to personalized medicine. N. Engl. J. Med. 363, 301–304 (2010).
Puhr, S., Calhoun, P., Welsh, J. & Walker, T. The effect of reduced self-monitored blood glucose testing after adoption of continuous glucose monitoring on hemoglobin A1c and time in range. Diabetes Technol. Ther. 20, 557–560 (2018).
Meuwese, C., Stenvinkel, P., Dekker, F. & Carrero, J. Monitoring of inflammation in patients on dialysis: forewarned is forearmed. Nat. Rev. Nephrol. 7, 166–176 (2011).
Della Ciana, L. & Caputo, G. Robust, reliable biosensor for continuous monitoring of urea during dialysis. Clin. Chem. 42, 1079–1085 (1996).
Nagler, R. M. Saliva analysis for monitoring dialysis and renal function. Clin. Chem. 54, 1415–1417 (2008).
Pickering, J. W. et al. Rapid rule-out of acute myocardial infarction with a single high-sensitivity cardiac troponin T measurement below the limit of detection: a collaborative meta-analysis. Ann. Intern. Med. 166, 715–724 (2017).
Hovorka, R. Continuous glucose monitoring and closed-loop systems. Diabet. Med. 23, 1–12 (2006).
Baker, D. A. & Gough, D. A. A continuous, implantable lactate sensor. Anal. Chem. 67, 1536–1540 (1995).
Khan, Y. et al. A flexible organic reflectance oximeter array. Proc. Natl Acad. Sci. USA 115, E11015–E11024 (2018).
Ferguson, B. S. et al. Real-time, aptamer-based tracking of circulating therapeutic agents in living animals. Sci. Transl. Med. 5, 213ra165 (2013).
Arroyo-currás, N. et al. Real-time measurement of small molecules directly in awake, ambulatory animals. Proc. Natl Acad. Sci. USA 114, 645–650 (2017).
Navaneelan, T., Alam, S., Peters, P. A. & Phillips, O. Deaths involving sepsis in Canada. Health at a Glance (21 July 2016).
Garg, S. K., Rewers, A. H. & Akturk, H. K. Ever-increasing insulin-requiring patients globally. Diabetes Technol. Ther. 20, S21–S24 (2018).
Diabetes: Key Facts (World Health Organization, 2018); https://www.who.int/news-room/fact-sheets/detail/diabetes
Basu, S. et al. Estimation of global insulin use for type 2 diabetes, 2018–30: a microsimulation analysis. Lancet Diabetes Endocrinol. 7, 25–33 (2019).
Casparie, A. F. & Elving, L. D. Severe hypoglycemia in diabetic patients: frequency, causes, prevention. Diabetes Care 8, 141–145 (1985).
Cryer, P., Davis, S. & Shamoon, H. Hypoglycemia in diabetes. Diabetes Care 26, 1902–1912 (2003).
Geller, A. et al. National estimates of insulin-related hypoglycemia and errors leading to emergency department visits and hospitalizations. JAMA Intern. Med. 174, 678–686 (2014).
Guerci, B. & Sauvanet, J. P. Subcutaneous insulin: pharmacokinetic variability and glycemic variability. Diabetes Metab. 31, 7–24 (2005).
Gin, H. & Hanaire-Broutin, H. Reproducibility and variability in the action of injected insulin. Diabetes Metab. 31, 7–13 (2005).
Heinemann, L. Variability of insulin absorption and insulin action. Diabetes Technol. Ther. 4, 673–682 (2002).
Wilson, B. D., Hariri, A. A., Thompson, I. A. P. & Eisenstein, M. Independent Control of the thermodynamic and kinetic properties of aptamer switches. Nat. Commun. 10, 5079 (2019).
Tang, Z. et al. Aptamer switch probe based on intramolecular displacement. J. Am. Chem. Soc. 130, 11268–11269 (2008).
Munzar, J. D., Ng, A. & Juncker, D. Duplexed aptamers: history, design, theory, and application to biosensing. Chem. Soc. Rev. 48, 1390–1419 (2019).
Nakatsuka, N. et al. Aptamer-field-effect transistors overcome debye length limitations for small-molecule sensing. Science 362, 319–324 (2019).
Li, J., Janle, E. & Campbell, W. W. Postprandial glycemic and insulinemic responses to common breakfast beverages consumed with a standard meal in adults who are overweight and obese. Nutrients 9, 32 (2017).
Bantle, J. P. et al. Postprandial glucose and insulin responses to meals containing different carbohydrates in normal and diabetic subjects. N. Engl. J. Med. 309, 7–12 (1983).
ter Braak, E. W. et al. Injection site effects on the pharmacokinetics and glucodynamics of insulin lispro and regular insulin. Diabetes Care 19, 1437–1440 (1996).
Home, P. D. The pharmacokinetics and pharmacodynamics of rapid-acting insulin analogues and their clinical consequences. Diabetes Obes. Metab. 14, 780–788 (2012).
Freckmann, G. et al. Continuous glucose profiles in healthy subjects under everyday life conditions and after different meals. J. Diabetes Sci. Technol. 1, 695–703 (2007).
Meijer, H. E. H., Singh, M. K., Kang, T. G., Den Toonder, J. M. J. & Anderson, P. D. Passive and active mixing in microfluidic devices. Macromol. Symp. 279, 201–209 (2009).
Marschewski, J. et al. Mixing with herringbone-inspired microstructures: overcoming the diffusion limit in co-laminar microfluidic devices. Lab Chip 15, 1923–1933 (2015).
Paek, S. H., Lee, S. H., Cho, J. H. & Kim, Y. S. Development of rapid one-step immunochromatographic assay. Methods 22, 53–60 (2000).
Pollema, C. H., Ruzicka, J., Christian, G. D., Lernmark, A. & Lernmark, A. Sequential injection immunoassay utilizing immunomagnetic beads. Anal. Chem. 64, 1356–1361 (1992).
Chang, L. et al. Single molecule enzyme-linked immunosorbent assays: theoretical considerations. J. Immunol. Methods 378, 102–115 (2012).
McGrath, J., Jimenez, M. & Bridle, H. Deterministic lateral displacement for particle separation: a review. Lab Chip 14, 4139–4158 (2014).
Gomis, S. et al. Single-cell tumbling enables high-resolution size profiling of retinal stem cells. ACS Appl. Mater. Interfaces 10, 34811–34816 (2018).
Peiris, H. et al. Discovering human diabetes-risk gene function with genetics and physiological assays. Nat. Commun. 9, 3855 (2018).
Adolfsson, P., Parkin, C. G., Thomas, A. & Krinelke, L. G. Selecting the appropriate continuous glucose monitoring system—a practical approach. Eur. Endocrinol. 14, 24–29 (2018).
Wu, K. & Huan, Y. Streptozotocin-induced diabetic models in mice and rats. Curr. Protoc. Pharm. 5, 5.47.1–5.47.20 (2008).
Humulin R (Eli Lilly and Company, 2018); https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/018780s120lbl.pdf
Humulin N (Eli Lilly and Company, 2018); https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/018781s121lbl.pdf
Woodworth, J. R., Howey, D. C. & Bowsher, R. R. Establishment of time-action profiles for regular and NPH insulin using pharmacodynamic modeling. Diabetes Care 17, 64–68 (1994).
Kim, J., Campbell, A. S., De Ávila, B. E. F. & Wang, J. Wearable biosensors for healthcare monitoring. Nat. Biotechnol. 37, 389–406 (2019).
Munje, R. D., Muthukumar, S., Jagannath, B. & Prasad, S. A new paradigm in sweat based wearable diagnostics biosensors using room temperature ionic liquids (RTILs). Sci. Rep. 7, 1950 (2017).
Hao, Z. et al. Measurement of cytokine biomarkers using an aptamer-based affinity graphene nanosensor on a flexible substrate toward wearable applications. Nanoscale 10, 21681–21688 (2018).
Visser, E. W. A., Yan, J., Van IJzendoorn, L. J. & Prins, M. W. J. Continuous biomarker monitoring by particle mobility sensing with single molecule resolution. Nat. Commun. 9, 2541 (2018).
Goodwin, M. L., Harris, J. E., Hernández, A. & Gladden, L. B. Blood lactate measurements and analysis during exercise: A guide for clinicians. J. Diabetes Sci. Technol. 1, 558–569 (2007).
Graf, A. et al. Moving toward a unified platform for insulin delivery and sensing of inputs relevant to an artificial pancreas. J. Diabetes Sci. Technol. 11, 308–314 (2017).
Wiesli, P. et al. Acute psychological stress affects glucose concentrations in patients with type 1 diabetes following food intake but not in the fasting state. Diabetes Care 28, 1910–1915 (2005).
This research was supported by the Chan Zuckerberg Biohub, a Stanford Diabetes Research Center (SDRC) Pilot grant and the Transdisciplinary Initiative Program (TIP) from the Stanford Maternal & Child Health Research Institute (MCHRI). C.L.M. was supported by a NSERC Postgraduate Scholarship and Stanford Bio-X Bowes Graduate Student Fellowship. We thank B. Buckingham and R. Lal for their helpful discussions as well as N. Maganzini and I. Thompson for their review and edits of the manuscript. We thank the Stanford Nanofabrication Facility (NSF) for their cleanroom facilities and the Canary Center at Stanford for Cancer Early Detection for their biolayer interferometry instrument. We thank the Stanford Veterinary Service Center staff for their assistance with animal care and procedures.
The authors declare no competing interests.
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Supplementary methods, figures, video captions and references.
Separation, by DLD, of fluorescently labelled microbeads from blood cells and free fluorescently tagged antibodies.
Glucose and insulin beads passing through the detection window.
Control experiment showing that glucose beads only fluoresce in their specific region in the upper part of the detection window.
Control experiment showing that insulin beads only fluoresce in their specific region in the bottom part of the detection window.
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Poudineh, M., Maikawa, C.L., Ma, E.Y. et al. A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals. Nat Biomed Eng 5, 53–63 (2021). https://doi.org/10.1038/s41551-020-00661-1
Nature Reviews Drug Discovery (2021)