Biosensors are a new sensor type that detects specific substances. They allow for highly sensitive and selective detection of various analytes, including glucose, lactate, and cholesterol. Biosensors are promising healthcare technology, providing real-time monitoring, such as blood glucose levels, without invasive procedures. Wearable biosensor data help diagnose accurate treatment plans or preventive prescriptions by health and sports professionals. This tutorial suggests building a proof-of-concept of an enzymatic lactate biosensor, promising to monitor muscular fatigue in athletes.

BioWatch

DeVinci-Innovation-Center ⋅ 6 months ago

Context

Biosensors are analytical devices that utilize biological recognition elements to detect and quantify target molecules. The first biosensor was developed in the 1960s by Clark and Lyons, who measured blood glucose levels for the first time [2]. Since then, biosensors have undergone significant advancements and have been widely used in various applications, including healthcare, food safety, environmental monitoring, and drug discovery [3].

After glucose, lactate is a biomarker on the way to being effectively monitored in the coming years. Human in vivo studies has demonstrated conspicuous concomitances between muscle lactate and muscle fatigue [4-5]. Muscle lactate is a well-known and relevant biomarker of exercise-induced muscle fatigue [6-7]. In recent years, developing non-invasive or minimally-invasive biosensors for continuous lactate monitoring has raised strong interest in wellness and sports applications [8]. Lactate monitoring can help professional sportives to select adequate exercise according to their performances [9-10].

Skills & Opportunities

The research within the biosensing field requires a multidisciplinary approach that involves different branches of science such as chemistry, biology, and engineering.

The first skill learned through the tutorial is electrochemistry. Electrochemistry is the study of the relationship between electricity and chemical reactions. This project aims to measure an electrical signal image of the chemical reaction. The practical application of the various manipulations also enables the acquisition of general technical laboratory skills.

The second skill is electronic programming. This tutorial uses a programmable analog front-end (AFE) development board, the LMP91000. It provides a complete signal path solution between a sensor and a microcontroller, generating an output voltage proportional to the cell current. This project allows a first approach to an engineering development board.

Requierments

This project requires the following:

• Basic chemical knowledge,
• Notions in C++,
• An LMP91000 AFE from Texas Instruments
• Lactate Oxidase (LOx),
• PolyPyrrol (PPy)
• Sodium Dodecyl Sulfate (SDS) (Optionnal),
• A coil of pure platinum wire
• Lactic acid solution
• Phosphate Buffer Saline (PBS)
• Soda,
• magnetic stirrer,
• A 1ml electronic micropipette,
• A wash bottle,
• A 2ml Eppendorf tube,
• One 500ml beaker,
• crocodile clips,
• Iron wires for electronics.

Biosensor Principles

Immobilization Matrix

Biosensor functionalization consists of immobilizing the enzyme on a transducer surface [11]. The four main immobilization methods are (1) non-covalent adsorption and deposition, (2) physical entrapment, (3) covalent attachment, and (4) bio-conjugation. This tutorial involves physical entrapment. Entrapment consists of the enzyme inclusion in a polymer network [12].

Enzyme

In fabricating L-lactate biosensors, the most commonly used biological recognition elements are lactate dehydrogenase (LDH) and lactate oxidase (LOx) [13]. The enzyme catalyzes lactate oxidation to pyruvate in dissolved oxygen and forms hydrogen peroxide. Hydrogen peroxide is electrochemically active and can be either reduced or oxidized to give a current proportional to the lactate concentration [14]. LOx and LDH involve simple enzymatic reactions and allow considerably simple sensor design fabrication [15]. This tutorial involves LOx due to its relatively lower price.

Membrane

The outer membrane selector has two functions:

• to filter out interferents: these biomolecules can interact with the enzyme and distort the signal.
• to regulate the concentration of the target molecule in contact with the enzyme to avoid oxygen lack so as not to saturate the biosensor [16].

This tutorial does not deal with membrane design since the membrane. Its two functions are not necessary for in vitro testing. The membrane also tends to reduce the sensitivity of the biosensor.

1.1. Prepare the platinum wire: Cut the platinum wire to a 5-centimeter length.

1.2. Prepare the PBS solution: Dilute PBS powder in 1 liter of distilled water. Fill a wash bottle with the PBS solution prepared.

1.3. Clean the platinum wire: Dissolve 0.8g of solid soda in a 100 ml beaker of distilled water using a stirrer. Leave the wires immersed in the soda solution for 10 minutes. Remove the wire, clean it thoroughly using ethanol, and dry it at room temperature.

1.4. Prepare the polypyrrole solution: Weigh 0.4 g of PPy powder and add it to a glass beaker containing 20 mL of acetone. Stir the mixture for 30 minutes at room temperature until the PPy powder is fully dissolved. Add 80 mL of distilled water to the solution and stir for 30 minutes to obtain a homogeneous PPy solution.

1.5. Dip-coat the platinum wire with PPy: 1. Dip the cleaned platinum wire into the PPy solution and withdraw it slowly at approximately 2 cm/min for 2 minutes. Use as topwatch and ruler if no dip coater, or leave immersed motionless. Dry the wire at 60°C in an oven for 30 minutes.

1.6. Prepare the functionalization solution: Pour 2 µl of LOx in a 2 ml Eppendorf tube using a micropipette and 2 ml of PBS. Close and shake the tube vigorously to homogenize.

1.7. Immobilize lactate oxidase by entrapment: Pour 0.2ml of the functionalization solution onto the last 2 cm platinum wire to immerse the PPy-coated platinum wire. Leave to stand for 6-12 hours at room temperature. The enzyme will be entrapped within the PPy layer.

1.8. Wash the immobilized wire: Remove the wire from the lactate oxidase solution and wash it with PBS.

2. LMP91000 Programming

Electrochemical biosensor tests require a potentiostat. A potentiostat is a device, or generally, an electronic circuitry, that allows the application potential to an electrode, called the working electrode (WE) [23]. By applying the potential of the target molecule oxidation (+650mV for lactate) or reduction potential, the enzyme catalyzes the molecule's oxidation. The reaction produces an or several electrons. The resulting current passes through the WE to the potentiostat circuitry and a counter electrode (CE). The potentiostat has AOPs that amplify the signal measured by order of microamperes. Chronoamperometry (CA) is an electrochemical technique that applies a fixed electrical potential at the WE and measures the intensity over time [24]. CA usually requires a calibration time of around one hour until the potential between the WE and the CE stabilizes. The current measured is proportional to the lactate concentration at the WE surface. The LMP91000 is a popular potentiostat board for micro-power electrochemical sensing applications [25].

3. Experimentation

3.1. Connect the electrodes: Connect the functionalized platinum wire to the working electrode of the LMP91000 with an alligator clip. Cut and connect two other platinum wires to the counter electrode and the reference electrode in the same manner. Check the connections with a voltmeter.

3.2. Setup the electrochemical cell: Fill a clean beaker with 400ml of PBS. Shake the cell constantly with a magnetic stirrer at medium speed.

3.3. Place the electrodes: Insert the platinum wires into the electrochemical cell, start the chronoamperometry code. Check that the values are consistent. Wait 3600 seconds.

3.4. Inject lactate: Add 160µL of lactic acid with a propette, to obtain a concentrated solution of 4mM. Repeat the procedure 4 times every 5 minutes, i.e. until a concentration of 20mM is obtained.

3.5. Exploit the results: Copy the data from the console (select all with Ctrl + A) and paste it into an Excel spreadsheet. Split the time and current values into two columns with the separator ",". Change the full stops to commas.

3.6. Characterize the sensor: Draw a graph of the inputs versus time. Identify the injections and calculate the sensitivity and linearity of the sensor.

Conclusion

This tutorial proposes to design a simple lactate biosensor from scratch. It involves the most accessible facilities possible. The biosensors manufactured and tested show poor performance. One possible interpretation is that the non-optimized wiring allows for strong signal perturbations. Future work will involve testing these with a commercial potentiostat. In addition, the platinum wire could be replaced by micro-needles painted with platinum paste. These micro-needles could then be integrated into the BioWatch.