A reagent-free paper-based sensor embedded in a 3D printing device for cholinesterase activity measurement in serum
Graphical abstract
Introduction
Cholinesterase represents a family of enzymes, present in the central and peripheral nervous system, in the muscles, and in erythrocytes, able to hydrolyze choline esters. Acetylcholinesterase (AChE) is localized both in peripheral nerves and in central nervous system, where it rapidly hydrolyzes the neurotransmitter acetylcholine for the termination of impulse transmission at cholinergic synapses [1]. Butyrylcholinesterase (BChE) is localized in muscle, in brain, and in other tissues [2]. The biological role of BChE is unknown, reason for that it has been considered an “orphan enzyme” with no specific physiological role, even if a recent study demonstrated that BChE hydrolyzes the so-called “hunger hormone”, which affects fat metabolism [3]. BChE was studied by pharmacologists because it is involved in the hydrolysis of succinylcholine, used in anesthesia as a short-acting blocker of acetylcholine receptor [4]. The physiological BChE activity is usually comprised between 5.9 and 13.2 IU/mL [5], and high BChE levels in serum are linked with some pathologies, such as obesity, diabetes, uremia, hyperthyroidism, and Alzheimer’s disease (AD), while decreased levels can indicate liver pathologies, such as acute hepatitis or liver cirrhosis [6]. Since the high level of BChE is able to transform the beta-amyloid in a malignant form, causing brain tissue degeneration, i.e., AD [7], [8], in 1993 the U.S. Food and Drug Administration approved the Cholinesterase inhibitors that today are the most common agents used for AD treatment. In the field of AD treatment, some studies recommend to monitor the patient response to the treatment, which can last up to three weeks [9], because the collateral effects during single or multidose drug infusions are not negligible (i.e. nausea, vomit, sweating, dizziness, headache, nightmares, and generalized fatigue).
A reliable device able to achieve a rapid, cost-effective, and affordable BChE activity measurement could then be very useful. The interest of many scientists has been recently focused on the development of integrated platforms for the autonomous (multi)monitoring of biological parameters.
A new promising trend is the use of eco-friendly paper-based Point Of Care (POC) devices. In this research field, Whitesides and co-workers already developed many naked-eye colorimetric assays, which are able to detect different biomarkers (e.g. glucose and protein) in complex matrices, such as serum and urine [10], [11]. One of the first paper-based electrochemical sensors was developed by Henry and co-workers [12], which designed a hydrophobic area on filter paper by photolithography and loaded it with several reagents (i.e., enzymes) to detect glucose, lactate, and uric acid. Actually, most patterns are made by wax printing because it is the most efficient and inexpensive way to fabricate microfluidic paper-based analytical devices (μPADs). These electrochemical devices printed on paper represent a sustainable low-cost technology; furthermore, they can be tuned in porosity, thickness and surface area by changing the paper grade: for example, Vella et al. used different types of paper for filtering red blood cells [13].
Another interesting technology which is emerging as a powerful tool for the fabrication of custom-made devices for analytical applications is the 3D printing. Some examples include purification systems, electronic sensors, injection valves, and supports for detection by smartphone [14], [15].
Although today a lot of POC devices have been proposed, the ones already available on the market are very few, e.g., glucose biosensors and naked-eye pregnancy tests. Indeed, in order to be suitable and successful on the market, such sensing devices need to accomplish several issues, including simplicity, affordability, portability, stability, reliability, and repeatability, which are fundamental requirements for both producer and end user.
Herein, we report the first example of a paper-based sensor embedded in a 3D printing device applied for the measurement of BChE activity in serum. The 3D printing device was fabricated with stereolithography technologies, because they offer the advantage to produce inexpensive transparent microfluidic devices with high precision.
The amount of BChE activity was estimated by quantifying the thiocholine enzymatic product in amperometric mode. Because at the bare electrode the detection of thiocholine requires a high applied potential that poses problems as fouling and interference, a nanocomposite made by Carbon Black and Prussian Blue was used as ink-nanomodifier to overcome these drawbacks. Furthermore, the porosity of filter paper was exploited to load the enzymatic substrate and the phosphate buffer to obtain a “reagent free” device for BChE detection.
Section snippets
Reagents and equipments
Potassium ferricyanide, ferric chloride, potassium hydroxide, hydrochloric acid, butyrylcholinesterase from equine serum, butyrylthiocholine chloride (BTCh), and potassium chloride were purchased from Sigma Aldrich, USA. Commercial carbon black (CB) N220 powder was received as a kind gift from Cabot Corporation, Italy. Graphite based ink (Electrodag 421), silver/silver chloride ink (Electrodag 4038 SS) were purchased from Acheson, UK. Cyclic voltammetric and chronoamperometric measurements were
Results and discussions
In this work, we reported the development of a paper-based and reagent-free sensor for the detection of BChE activity in serum. With the aim to develop a selective and sensitive sensor, the first section of the experimental work was dedicated to optimize the working conditions in standard solutions to seek the linear range and detection limit useful to apply this sensor in biomedical field. As soon as a tailored configuration was obtained, the paper-based sensor embedded in a 3D-holder was
Conclusions
In this work we demonstrated the advantages to combine i) a printing technology to develop a mass produced sensor, ii) a 3D printing to easily customize a holder for the sensor, iii) wax printing to fabricate microfluidic patterns in a sustainable way, iv) the use of paper as eco-friendly substrate to print the electrochemical cell for a reagent free device. The use of the paper is further advantageous in the case of sensors tested with biological samples, because, after the measurement, the
Giorgio Scordo is graduated in Biomedical Engineering. His research interests include the 3D printing, nanomaterials, and electrochemical sensors.
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Giorgio Scordo is graduated in Biomedical Engineering. His research interests include the 3D printing, nanomaterials, and electrochemical sensors.
Danila Moscone is full Professor of Analytical Chemistry. Prof. Moscone’s activity concerns the construction of different biosensors and its application in analytical matrices since thirty years. During this time, she improved her experience in the field of electrochemical sensors and flow systems coupled to biosensors. The research activity carried out was published in several papers as follows: 30 chapters on books, 2 reviews, 1 monograph, 137 papers on international and national scientific journals, 2 patents, 36 proceedings, 2 videos, more than 300 oral and poster presentations at scientific meetings.
Giuseppe Palleschi is full Professor of Analytical Chemistry, has been the Head of the Department of Chemical Science and Technology of the University of Rome Tor Vergata for 1995–2007. In 2000, he obtained the “Laurea Honoris Causa” from the University of Bucharest. Prof. Palleschi’s research over the last 30 years has been focused on the development of chemical sensors as well as bio- and immunosensors for use in the areas of biomedicine, food and environmental analysis. He is the author of more than 280 papers in international scientific journals and an invited speaker in many International Congresses.
Fabiana Arduini is currently Senior Researcher at Department of Chemical Science and Technology (Analytical Chemistry), University of Rome Tor Vergata. Her research interests include the development of Electrochemical sensors, Electrochemical Biosensors, Screen-Printed Electrodes, Sensor system modified with nanomaterials. She works on real applications in the field of environmental, food, and clinical analytical chemistry. Her research activity has been published in several papers as follows: 64 articles (9 reviews) in ISI peer-reviewed journals, 11 chapters in books, 5 proceedings, 22 articles as first-author + 39 articles as corresponding author. Her H-index is 25, with 1761 total citations (Scopus, July 2017).