CHAPTER 3. Fully integrated lab-on-a-disc for determination of natural antioxidants from
3.2 Available methods for total phenolic content and antioxidant activity
Analytical methods for determination of total phenolic content and antioxidant evaluation in different types of sample with analysis time required are shown in Table 3.1. Conventionally, total phenolic content and antioxidant activity detection were conducted using chromatographic methods; (GC)135 and high performance liquid chromatography (HPLC)136 or using colorimetric methods. Both GC and HPLC analyses are powerful for separating and identifying the total phenolic content components in the complex sample but both required complicated sample preparation, long analysis time and expensive operating machine. Therefore, both GC and HPLC methods are not practical for routine analysis in point of care test settings.
The traditional colorimetric method is frequently used for determination of total phenolic content and antioxidant activity because of its easier availability in many laboratories. A Folin-Ciocalteu (FC) method is commonly used for determination of the total phenolic content in plant extracts and beverages.128 The most common methods widely used for evaluation of antioxidant activity in food products and dietary supplements are based on radical scavenging, such as 2,2-diphenyl-1- picrylhydrazyl radical (DPPH),61 trolox equivalent antioxidant capacity (TEAC)137 assay, and ferric reducing antioxidant power (FRAP) assay.138 These colorimetric methods are simpler than conventional GC or HPLC analysis but it also requires
Table 3.1. Analytical methods for determination of total phenolic content and antioxidant evaluation in different types of sample with analysis time required.
Method Sample Analysis time
Total phenolic content
GC–MS135 Moringa peregrine
(plant) 70 min
HPLC–UV136 Grape juice 60 min
Spectrophotometer139
(based on FC reaction) Commercial beverage 90 min Flow injection analysis63
(based on FC reaction) Food product 5 min Sequential injection analysis64
(based on FC reaction) Wine 5 min
Lab-on-a-chip3 (based on acidic potassium
permanganate chemiluminescence)
Honey 10 min
Antioxidant activity Spectrophotometer61
(based on DPPH reaction) Food 30 min
Spectrophotometer138
(based on FRAP reaction) Teas 30 min
Spectrophotometer140
(based on TEAC reaction) Vegetable juices 30 min SIA141
(based on ABTS reaction) Ginger beverages 1.5 min Lab-on-a-chip6
(based on DPPH reaction) Nutrients 10 min
time consuming steps and labor-intensive manual handling. Additionally, recent studies claimed that it is necessary to combine more than one method to measure antioxidant activity in beverage samples since the antioxidant activity of food and beverage samples are determined by a mixture of different antioxidants with different mechanisms of actions. Therefore, the automation and integration of more than one detection methods are required to avoid many labor-intensive manual handling and long reaction time.
To automate method base on colorimetric reaction, flow injection analysis (FIA)63 and sequential injection analysis (SIA)64 have been developed for determination of total phenolic content of wine and beer samples. These methods have novelty in reducing the time, labor and handling errors from manual experiment. However, it required complicated pumping and tubing in FIA, and it required large sample and reagent volume in SIA. Also, both methods did not measure both total phenolic content and antioxidant activity.
Recently, lab-on-a-chip system has been employed for a simple one-step reaction and detection of total phenolic content and antioxidant activity in microfluidic channels. It has been developed for determination of total phenolic content in honey3 and a lab-on-a-chip device has also been developed a two-compartment microfluidic device that minimizes the dynamic of liver metabolism and the subsequent antioxidant activity of food components by Jungwoo Lee and coworkers.6 They used 2,2- diphenyl-1-picrylhydrazyl radical (DPPH) method for antioxidant activity detection. Both lab-on-a- chip based total phenolic content and antioxidant activity detection have novelty in automating the complicated steps and reducing the sample and reagent volume. However, this technique requires external syringe pumps and external interconnects to induce fluid movement. In addition, both studies cannot detect total phenolic content and antioxidant activity simultaneously, and also in Lee’s work, they only used DPPH method for antioxidant activity detection.
Centrifugal microfluidic system called lab-on-a-disc has garnered much attention as analytical device for automating and integrating complicated assay in variety field such as clinical,86 environmental,5 chemical engineering14 and food application.2 In lab-on-a-disc system, a simple motor is used to rotate the microfluidic disc device and then, centrifugal force is the driving force for fluidic or particles transported through microchannels. By controlling fluid direction using valves, all process from sample metering to detection could be integrated and automatically conducted on a rotating disc device.106, 142,
143 As taking advantages describe above, lab-on-a-disc can be an ideal solution to overcome the limitation of previous total phenolic content and antioxidant activity detection such as huge consumption of reagents, requiring complex pumping, expensive cost, long analysis time and labor intensive manual handling, and minimal amount of instrument.
In this work, we present a new fully integrated lab-on-a-disc system for simultaneous determination of total phenolic content and antioxidant activity detection from different beverage samples (teas, fruit juices, beer and wines). Lab-on-disc platform was designed and developed the all-in-one device for three different colorimetric detections including FC, DPPH and FRAP methods. The optimization of sample and reagent volume consumption, mixing time of three colorimetric reaction (FC, DPPH and FRAP) and sample dilution ratio for the integration on a disc were investigated. The full automation of all steps such as sample filtering, metering, mixing, reaction and final colorimetric detection was demonstrated. The lab-on-a-disc method was validated with the conventional method for total phenolic content and antioxidant activity analysis in different beverage samples.