Analytica Chimica Acta 676 (2010) 68–74 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Nanoengineered optical urea biosensor for estimating hemodialysis parameters in spent dialysate M. Swati a , N.K. Hase b , Rohit Srivastava a,∗ a Department of Biosciences and Bioengineering, Centre for Research in Nanotechnology & Science (CRNTS), IIT Bombay, Powai, Mumbai 400076, Maharashtra, India b Department of Nephrology, King Edward Memorial Hospital, Parel, Mumbai 400012, India article info Article history: Received 8 April 2010 Received in revised form 16 June 2010 Accepted 20 July 2010 Available online 27 July 2010 Keywords: Hemodialysis monitoring Optical urea biosensor Urea kinetic modeling Calcium alginate microspheres Cresol red Layer-by-layer abstract An optical biosensing scheme based on urease encapsulated calcium alginate microspheres which are coated with polyelectrolyte nanofilms predominantly composed of cresol red (CR) dye is demon- strated in this paper. The dye molecules within the nanofilms are deposited via the layer-by-layer (LbL) self-assembly technique on the microspheres and used as the optical transducer. A flow through cell con- structed using a cuvette attached to a fiber optic spectrometer was used to determine the response of the biosensor to standard urea solutions of different concentrations. The change in pH and the absorbance ratio was monitored with time and these results were used for measurements of urea concentrations in the spent dialysate fluid. The biological parameters controlling hemodialysis such as dialyzer clearance or Kt/V and percent removed urea (PRU) have also been reported. The results demonstrate that the urea biosensor is pH reversible with a sensitivity of 0.09 pH units/min and is able to detect a change of 0.005 ratio units in urea concentration ranging 0.1–60 mg dL -1 . The response time of the sensor was calculated as 8 min while the detection range of urea covered the levels that are present in the spent dialysate fluid. The results obtained in the analysis of biological samples were in good agreement with those obtained by a reference method, showing no significant differences at a confidence level of 95%. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Dialysis is necessary to remove waste products from the blood, when kidneys fail to function. Nearly all uremic toxins are prod- ucts of protein metabolism and clinically it is difficult to identify and determine all of them. While whole spectrum of these prod- ucts is unknown, urea (having mild toxicity) has become the marker for these unidentified toxins because it is the end product of pro- tein metabolism and is present in millimolar (mM) concentration which is easy and relatively inexpensive for determination [1]. Dial- ysis efficiency is monitored periodically by testing a patient’s blood which is sampled at the start and at the end of dialysis. The levels of urea in the two blood samples are then compared. Urea kinetic modeling [2–7] is increasingly recognized as the most efficient way for quantifying and monitoring hemodialysis treatments. Mathe- matical models that have been applied for urea kinetic modeling are complex, time-consuming, require blood sampling and potentially inaccurate. Direct dialysate methods bypass any solute disequilib- rium in the patient’s body. This simplifies the mathematics and allows accurate predictions to equilibrium concentrations in the body, even before all solute gradients have dissipated [8]. ∗ Corresponding author. Tel.: +91 22 25767746; fax: +91 22 25723840. E-mail address: rsrivasta@iitb.ac.in (R. Srivastava). Measurement of urea in the spent dialysate makes it possible to calculate the amount of urea removed during one session directly and is termed as true removed urea (TRU). This value is highest at the beginning of the week and lowest at the end of the week. The bioanalytical systems dedicated for dialysate urea nitrogen (DUN) determination predominantly are based on enzymatic reactors containing urease. The enzyme converts urea into ionic prod- ucts (ammonium and hydrocarbonate/carbonate ions). Usually, for detection of the products, conductimetric [9,10] or potentiometric [11,12] techniques are adopted. For example, disposable conducti- metric biosensors for measurements in spent dialysate have been developed using screen-printing technology [13,14]. These biosen- sors for measurement of urea in spent dialysate have one of the major disadvantages that their fabrication takes long time with the requirement of expensive reagents at several steps. Even though they display faster response time, the range of urea levels detected by such sensors is usually narrow which might not be helpful in dealing with biological samples. Two methods are generally used to assess dialysis adequacy, percent removed urea (PRU) and Kt/V where K stands for the dialyzer clearance (mL min -1 ) and t stands for time. Kt/V is the fraction that represents the vol- ume of fluid completely cleared of urea during a single treatment (www.kidney.niddk.nih.gov accessed on 14.07.09). At present, there are some commercially available urea moni- tors for the automatic measurement of urea concentration in the 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.07.030