BİOSENSORS APPLICATIONS ON ORGAN ON A CHIP

Background

Organ on a chip is a microfluidic system which aims to recreate organ models or tissues in a small scale. This technology is mostly used on the drug development field and has been a milestone on the rise of personalized medicine. One of the biggest objectives related to this technology is the replacment cell cultures and usage of the animals for studying homologus illnesses on the humans. Lots of organ on chips have been fabricated and several of them are designed for multiorgans studies. There are still limitations on the integration of the biosensors needed for the continual measurement of microenvironmental parameters and control of the dynamical behaviors of the organ on the chips toward the different drugs tested. Real time biosensors on the other hand are crucial about achieving the automation of in situ monitoring of the different biophysical and biochemical parameters of the organ on the chips [1].

Keywords: organ on the chip, microfluidics, biosensors

Introduction

The cell cultures and the animal models for experimentinf the different drugs are limiting methods and they are very expensive. Also several human illnesses cannot be studied in those systems as they lack homology in animals. This yields to the necessity of more similar models to the human organs and practically a new system has been developed:the organ on a chip [2].

Figure 1: A scheme representing the different parts integrated on an organ on a chip.

State Art Technologies

Biosensors based Organ on a Chip for Drug Development

Several organs on the chips have been developed for testing different drugs. Most of those organ on the chips have been unsuccessful in achievment of a real time control because of a failure in the integration of the biosensors. Some prototypes have been buildt about measurement of glucose and lactate, oxygen level, the ions, acidification rate of the extracellular environment, pH, protein biomarkers and transepithelial resistance created by the electrical charges on the medium. Those prototypes did not intend to realize a full measurment or continuity and usually managed the measurement of a single signal for several hours. The major problem in the integration of the biosensors on the organ on chips is their incompatibility [2]. Recently, one biosensor-based-organ on the chip was manufactured where physical biosensors (O2 and pH measurement), microfluidic breadbord (timing the route of the fluids on the organ on a chip), electrochemical biosensors (biochemicals measurement) and minute microscopes were integrated. Benchtop incubator kept the level of CO2 and the temperature in a costant level. Nitrogen powered this model of organ on a chip by application through Wago Controller and Festo Valves and some MATLAB codes, which were also responsible for controlling the electrochemical station. Physical biosensors were controlled by a data acquisiter that was coworking with a LAbView program. In this sytem, a flow biosensor was integrated about monitoring the leakage, potential blockage and the flow rate over the system [3]. Fluids flow was realized by interconnection made by Teflon tubes. Interference of the biosensors with the other parts of the organ on a chip was prevented by a device that captured the air bubbles and removed from the system. This feature did not only improve the performance of the biosensors, but also provided a clearer image of the organ’s morphology[4]. The sensitivity and the real-long time ability of monitoring of those biosensors determine whether the integration on organ on a chip is successful or not [5]. The generation of an electrochemical biosensor was realized by coating a gold microelectrode with a self assembled monolayer by using 11-MUA [6] and immobilizing the streptavidin over it through a carodilimide reaction. This bonding provides the binding of the antibiodies in a stable manner. On the other hand, the measurement is realized due to the redox probe’s electrotransfer kinetics. The concentration im the solution and the amount of biomarkers captured on the probe are proportional to each other. Several biomarkers could be measured due to the integration of the immunobiosensor in the organ on a chip. Liver on a chip and heart on a chip were manufactured by the same scientific group in order to make practical measurements of the efficacy of the integration of the biosensors. The liver biomarkers, such as albumin and Gluthathione S-Transferase and the heart biomarker such as creatinine kinase MB were chosen to be measured continuously. Gluthathione is an important biomarker about detection of formation of reactive metabolites, while albumin is important for measuring the oxidative stress. On the other hand, the creatinine is a crucial biomarker in detection of several renal-cardiac disorder [7]. The performances of the immunobiosensors to detect and measure those specific biomarkers were recorded and calibration curves for each of them were obtained. The results were very promising as the three biomarkers were measured with a high sensitivity rate, specifically: 1.607, 1.105, and 1.483 log(ng·mL−1 ) −1. GST, CK-MB and albumin biosensors responded only to the biomolecule they were designed to measure. On the other experiments, a single immunobiosensor where several biomolecules could be measured was designed for the organ on a chip. Liver on a chip biosensor was fabricated where biomarkers, such as antitrypsin, ceruloplasmin and transferin could be measured continuosly on the same time. Also troponin detecting biosensors were fabricated for heart on a chip. The stability and sensitivity of those biosensors were increased by usage of the aptamers that have a higher specificity toward the antigens and are more stable on different

conditions. The impedance biosensors that were manufactured in this case measured the charge transfer occuring between electrode and the bioreceptors. pH on the organ on a chip was measured by an optical biosensor which could detect the absorption of the medium where phenol red was also present. The optical pH biosensor had a sensitivity of 0.159 V·pH−1 and was calibrated at media which differed on the pH value. Oxygen level was measured by an optical biosensor whose principle was the oxygen senstivity toward the ruthenium dye’s sensitive fluorescence. Its characterization was studied on media where different concentrations of nitrogen and air were present. According to the data recorded on this experiment, the oxygen biosensor was able to measure the changes in oxygen level very fast. The graph yieldt a linear regression where biosensor’s sensitivity was 7 mV·O2%−1. The reason that an optical biosensor was used to measure oxygen level was its suitability about online monitoring. The temperature biosensor based on the benchtop showed good results over a week measurement [1]. Long term monitoring of the chronic drug responses were also measured. Effect of a thymiilate synthase inhibitor (capecitabine) was monitored on both the liver on the chip and heart on the chip where several biosensors were integrated. The capacity of the integrated biosensors for detection of toxicity caused by different doses of acetaminophen (APAP) was continuosly measured. The pH, oxygen and temperature biosensors remained stable even after the application of different drug doses. The stability of oxygen biosensors is thought to have been caused by the gas permeability that PDMs devices have (PDMS substrate is usually used on the organ on a chip). This view is supported also by the experiment data that show that the perfusion’s flow rate is low, specifically 200 μL·h−1. On the other hand, the immunobiosensors measurements of the biomarkers, respectivally albumin and gluthathione showed difference on the levels of the biomarkers in a dose dependent fashion. CK-MB level on the Heart-on-a-Chip showed only slight changes during dose applications. CK-MB major changes were recorded from the immunobiosensor during the application of the drug DOX. Several limitations were still present on the system developed by scientists. One of them is related to the usage of PDMS apart from the fact that is has ability to adsorbe different drugs and materials present on the organ on a chip. This adsorption can lead to several mistakes on the measurement. DOX while applied on high concentrations was adsorbed from PDMS during the experiment. Development of thermoplastic microfluidics can solve this problem in the future applications. There remains hope due to this experiment’s positive sensitivity values that the futher experiments are going to realize more sensitive and stable, full automated multibiosensor integrated multiorgan on chips [7].

Figure 2: Supporting material from this experiment. The red arrows on the G-J demonstrate the times where the dose of the drug was added on the organ on a chip.

Label-Free & Regenerative Biosensors

Ability in detection of minute amounts of the biomarkers and continuous real time monitoring for a long time remain still challenging in the field of organ on a chip. The biosensors that are generally used are: ELISA, fluorescence based detection, Mass Spectrometry, SRPS and electrochemical detection, but they do not garantuee a total saturation of the tartget molecules while bound. This yields to inaccurate and noncontinuous measurements of the biomarkers. Probe regeneration is another problem, because of its necessity for the total interface to be reconstructed. This process is long in time and difficult. Sensitivity is also a problem related to the general biosensors used in organ on a chip as their surface gets damaged during the regeneration [8]. Regenerated biosensors have been developed by different cleaning processes about increasing the sensitivity of organ on the chips. The problem with those biosensors is that they are practically unable to measure the biomarker continuously. The ideal biosensor for an organ on a chip needs to be durable toward the cleaning processes and should last for several days continuously. Another feature of the biosensors that is crucial about their integration in the organ on the chip is their scalability about monitoring several biomarkers. On the other hand, it should be compatible with the bioreactor platforms and it should comprise only a small volume on the organ on a chip [9]. Electrochemical biosensors are the most preferred biosensors for being used on the organ on the chip due to their extraordinary limit of detection, ability for label free detection, portability, durability on long term, a wide response range and simplicity. It is easy to achieve the specific binding and increase the detection rate of the biomolecules by simply adding antibodies on the electrochemical biosensors (EC). Also ECs are ideal for integration on the organ on the chip as they can be easily miniaturized. The classic method for the measurements is ELISA and it is not suitable for the measurements on the organ on the chip as the continuous changes of the cellular responses cannot be measured [10]. A group of scientists developed a regenerative electrochemical biosensor and integrated it on a liver on a chip for measuring the hepatotoxicity. The biomarkers, such as gluthathione S-transferase and albumin were measured. The acetaminophen drug was used about causing hepatotoxicity. The accuracy of the biosensor was used by comparing the results of the same experiment by using ELISA. Electrochemical biosensor consists of 3 electrodes, respectivally: silver reference electrode, gold based working electrode and gold based counter electrode. The gold was chosen as a material for the electrode due to its stability, conductivity, biocompatibility, suitable electron kinetics and ability for good covalent bond formation. The adhesion of the Au and Ti layer that was added on the etching process was improved by putting a palladinium layer between them. Pa layer enabled the organ on the chip system to be secure from the extreme pH values, high electricity applications and usage of corrosive solvents. Alignement of the antibodies was improved by usage of a self assembled monolayer by the assistance of 11-mercaptoundecanoic acid and later by immobilization with streptavidin. The fluid flow from the bioreactor make the receptors leave the surface of the microelectrodes and this was prevented by the realization of the interaction between streptavidin and biotin. Sensitivity of the electrochemical biosensors can be regulated by optimizing several parameters. Incubation of them in complex media may provides with the sensitivity value. Generally, complex media can give bad results as it may include nonspecific proteins. The measurements about specificity were conducted on cell culture media consisting of fetal bovine serum (99 mg mL−1 ) [11]. The electrochemical biosensor was able to measure the albumin with a limit of detection of 0.023 ng mL−1 and with sensitivity of 0.95 (log(ng mL−1))−1. From the values of the experiment, it could be observed that the LOD value was lower than the one in the case an impedance based biosensor was used [1]. As SPV generally enables the antibody to orientate on the surface of the elctrode, the antigen binding ability is expected to be improved in this experiment. In my opinion, the reason why the LOD was different in those 2 experiments

whose only difference is the type of the biosensor, is because the impedance is less destructive than amperometry due to the long range of frequencies. The amperometry on the other hand is more preferred as it gives more precise values. In this experiment the sensitivity values (albumin detection) were higher than in the case where the impedance based biosensor was used [10]. The selectivity of the biosensor was measured by introducing it with different biomarkers of different concentration. The specific antigen was the GST-alpha which is produced in case liver gets damaged. On the other case, the albumin and CK-MB were used as nonspecific antigens in this experiment as they are produced in the case the cardiac tissue is damaged. The biggest challenge this biosensor has to overcome is that its electrodes rapidly saturate therefore its durability is shortened. That is the reason it is do dependable upon the regeneration process. The EC biosensor showed better results compared to the ELISA in accuracy and its miniaturization was more suitable for the organs on the chip. Also its lower LOD and beter sensitivity value make it promising for future applications.

Table 1: The values expressing the differences in sensitivity, LOD, accuracy, the range of detection, and the volume of the samples between ELISA and electrochemical biosensor used in this experiment.

Sensitivity LOD (ng/ml) Accuracy The range of detection (ng/ml) Volume of the sample (μL)

ELISA 1.5 ng/ml-1 0.2 8.7 3.125 – 200 50

EC

Biosensor 1.35 log(ng/ml)-1 0.09 9.6 0.1 – 100 7

On the other hand, the concentration of the GST-alpha was found to increase after the exposure to the drug APAP, while the level of the biomarker albumin was found to decrease. The values of the biomakeres were similar whether measured by EC biosensor or by ELISA. In my opinion the system those scientists have tested during this experiment has several advantages, such as full automation, detection in the mode by free antigens, regenerative ability, low cost, minute size and its superiority toward the earlier methods which have a problems in disentigration with the organs on the chips [7].

Figure 3: A representative image of the most important results of the experiment related to the state art I am briefly explaining. A)The hepatocyte images of the sealed and the primary one. B-C)The measurements about the albumin and GSt-alpha respectivally in different exposion values to the drug. D)Staining picture of the living and the dead hepatocyte toward control group. E) Scheme of the biosensor integrated with organ on a chip. F) Measurements of the biomarkers in the absence of the drug.

Organic Electrochemical Transistors

Organ on a chip technology has revolutionized the organ study fields and till now several organ on chips have been developed, such as: lung on a chip, skin on a chip, multiorgans on a chip etc. There is still work about developing more complex and more innovative models of organ on chips and about integration of in line biosensors to monitor the cell metabolites and the microfluidic environment’s transepithelial resistance. There is also a demand for an increase in compatibility of biosensors, optical biosensors and those organ on chips. Several functions, such as cell differentiation, dynamics, proliferation etc are going to be directly affected. Bioeelctronics is a field that is striving to build beter biosensors for organ on chips and any vitro cell model [8]. Organic electrochemical transistors are devices consisting of 3 gates and can be used for processes, such as chemical sensing, biosensing, in vivo recording, measurement of cells’s electrical parameters, such as capacitance and resistance. This technology is in the same time highly compatible with optical systems, such as fluorescence microscope. Integration of those organic electrochemical transistors in organ on chips as sensing units is expected to be a milestone, because of the high sensitivity and specificity in measurement of different metabolites [9]. A model was fabricated under laboratory conditions where organic electrochemical transistors were integrated with a microfluidic system in order to study in vitro toxicology, where the laminar flow enables the rise of shear stress on the cells. Cell glucose uptake was realized by OECT that served both as a transducer and amplifier [10]. The importance of this OECT in the future of organ on the chips is due to the fact that nowadays the assesment of them occurs only at the end of the procedure. Simultaneous monitoring would be able if this technology is integrated with organ on a chip.

Future Perspectives

If the biosensors can be fully integrated in the organ on the chips, several other field of studies, apart from drug development would be revolutionized. Currently there are stil several problems related to the differentative process of the cell, regeneration of the cells and blood, inflammation, system failure and toxicity. Organ on the chips have been unable to integrate in fields, such as chronic illnesses, autoimmune diseases, complex endocrine systems and skeletal illnesses. This disintegration is caused by the lack of full integrative biosensors as well. In my opininon one of the biggest challenges about sensors disintegration is caused by the relation between organ on the chips and the fabricative materials. PDMS is the mostly used for fabrication method due to its optical clarity, gas permeability and its biocompatibility, but in several experiments it has been shown to absorb the organic materials, drugs and polymers when present in high concentrations. In my opininon PDMS has to be replaced by fabricating materials that do not absorb the materials transported on the fluids. Several experiments have replaced this material by using ECM coatings, but it has not been so promising. One of the biggest challenges that the biosensors integrated organ on the chips have to overcome is the technical robustness. The necessity for an universal blood substitute arises. Real time monitoring stil remains under scientific study because of a current inability for absolute (error free) continuous measurement.

REFERENCES

1.https://www.technologynetworks.com/drug-discovery/articles/organs-on-chips-applications-challenges-and-the-future-288031

2.Multibiosensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors. Available from: https://www.researchgate.net/publication/314273579_Multibiosensor-integrated_organs-on-chips_platform_for_automated_and_continual_in_situ_monitoring_of_organoid_behaviors [accessed Jan 14 2018].

3. https://www.nature.com/articles/nrd4539 retrieved from Google source on 16.01.2018

4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379057/

5. Inamdar, N. K. & Borenstein, J. T. Microfluidic cell culture models for tissue engineering. Curr. Opin. Biotechnol. 22, 681–689 (2011).

6. https://www.technologynetworks.com/drug-discovery/articles/organs-on-chips-applications-challenges-and-the-future-288031

7. Huh, D., Torisawa, Y. S., Hamilton, G. A., Kim, H. J. & Ingber, D. E. Microengineered physiological biomimicry: organs-on-chips. Lab Chip 12, 2006–2105 (2012).

8. Sackmann, E. K., Fulton, A. L. & Beebe, D. J. The present and future role of microfluidics in biomedical research. Nature507, 161–199 (2014).

9. Agarwal A., Goss J. A., Cho A., McCain M. L. & Parker K. K. Microfluidic heart on a chip for higher throughput pharmacological studies. Lab Chip 13, 3599–3608 (2013).

10. Hornick J. E., Duncan F. E., Shea L. D. & Woodruff T. K. Multiple follicle culture supports primary follicle growth through paracrine-acting signals. Reproduction 145, 19–32 (2013).

11. Zhang YS, Khademhosseini A. Seeking the right context for evaluating nanomedicine: From tissue models in petri dishes to microfluidic organs-on-a-chip. Nanomedicine (Lond) 2015;10(5):685–688.

LAB ON A CHIP

Background

Lab on a chip is a device of minute magnitude consisting of channels and wells conjugated over a silicon or polymer substrate. Fluids of nano/microscale based volumes move through the channels of the lab on a chip (LOC) due to the electrokinetic forces or pressure exerted. LOC-s can also be known as microfluidics. LOC is able to realize the mixing, handling dilution, chromatographic separation, detection and staining proces. Recently the lab on chip and microfluidics technology are used in other fields of technology, such as molecular biology, genomics, agriculture, healthcare and physics field.

Keywords: lab on a chip, microfluidics, healthcare, technology

INTRODUCTION

1.1 History of LOC

The first LOC was a gas chromatograph. After its development, the equipments, such as pressure sensors, valves, pumps and fluid based systems were produced. LOC manufacture started to fluorish after the development of the method of photolithography, which was similar to the semiconductance method [1].

1.2 Procedure

Photolithography and soft lithography are crucial methods on LOC development where the primary process is the photolithography. Photolithography enables the trasfer of a specific circuit in a substrate therefore building an integrated circuit. It also enables the creation of channels and wells about producing LOC. The initial step consists on creation of a network (channels and wells) in a computer program. A printout is taken afterwards and this is differently called mask. The mask is transferred on a substrate and usually a silicon substrate coated by a thin film and photoresist is preferrable. The light irradiation process is applied. The development process is applied about stripping off the photoresist portion which did not get hardened by irradiation. The thin film not attached to the photoresist is removed and this step is known as etching. This procedure can be realized by either application of plasma or ions (dry etching) or using chemicals (wet etching). The structure remaining can be converted on a closed microchannel by covering with a slide and finally the creation of some holes on the slide leads to the production of inlets and outlets [2]. Soft lithography is a more modern method in which a mold is done by photolithography. Polymer gel is poured and cured due to crosslinking technique. Finally a replica is done after the removal of the mold. The repetition of the process leads to the manufacture of a LOC. This technique is cheaper and more efficient thant the method of photolithography whose main element is silicon that is

considered a rigid material. The most crucial element used in soft lithography is the optical transparent PDMS (polydimethylsiloxane) and its elasticity to create pumps and valves [3].

1.3 Mixing in LOC

The mixing property is one of the most important properties in LOC. The molecules on LOC move similarly to the stream lines of a laminar flow. Movement perpendicularly to the flow is a necessity for the mixing process and the molecular diffusion is a process that makes it possible. Reynold number or the ratio of forces on the LOC, respectivally: viscosity and the length. A smalle value of Renold represents a flow whish is absolutely laminar. The LOC systems with extremely low Reynold value have problems caused by the poor mixing of reagents. Nowadays techniques such as pulse mixe, the shortening of dicrete plugs or building of the microchannel on a serpentine shape therefore achieving the occurrence of both perpendicular and axial diffusion [4].

1.4 Sample and Reagent Introduction

The solutions and reagents are introduced on the LOC’s microfluidic channels by the usage of microelectronic methods, such as application of high voltage on the channel or application of a micromachined pump which lead to the creation of electrosmotic flow. The electrosmotic flow and the pump mechanism of LOC enables the capillary electrophoresis. Nowadays methods, such as syringe and peristaltic pump respectivally. Modern LOCs intend the elimination of the pump’s necessity. The movement of the flow realized without the need of the pump can ocur when the the iner surface of the microchannel is made hydrophilic. This movment is known as capillary action [5]. The usage of compact discs where the fabricated microchannels are on the central upward part of the disc, would also be a good strategy [6].

2. Different types of LOCs

2.1 Paper based LOCs

Paper provides a good opportunity to build LOCs as it is a cheap, easily fabricated, thin and easily manipulatable material compared to silicon which was the initial material for building LOCs. The fabrication of paper based LOCs is known as paper microfluidics and the devices fabricated with that way are known as microfluidic paper analytic devices. The fabrication method resembles the photolithography method. The main difference in paper based LOC’s fabrication is that instead of the hollow microfluidic channel used in photolithography, the channel is full of paper [7]. This method is inappropriate to be used for viscous materials as the paper fiber prevents the free flow. However paper based method is ideal for process of filtration where chromatographic filter paper is the most efficient method. Several paper based assays about analysis of samples such as: glucose, lactate, alcohol, heavy metals, proteins, enzymes are being fabricated nowadays [7].

2.2 PDMS Lab on a Chips

LOC manufactured by using polydimethylsiloxane (PDMS) which is flexible, elastic and cheap material enables a fast flow and air permeability on the sample culture needed to be analyzed on the LOC. PDMS based LOCs have several disadvantages, because of the difficulty of integration of electrodes in the chip, aging process of PDMS and the adsorption of hydrophilic molecules by PDMS [8].

2.3 Thermopolymers LOCs

Thermopolymer LOCs are expensive and difficult to be manufactured, but they are highly used. The main component used in their fabrication is themoplastic which is a transparent material. It is preferrable for the lithography method due to its micrometric scale and is a more preferrable method than PDMS due its chemical inertness. Thermopolymers’s research provides hope for a large scale of LOC’s industrialization [9].

2.4 Glass LOCs

Those LOCs are characterized by transparance, micrometric scale and chemical interness. The glass lab on chips are advantagious as they can be easily industrialized, due to the easily integration of the reproducible electrodes and the wide range of the chemicals that can be used aboıut the treatment of its surface. The microfabrication of the glass LOCs requires specialized researchers and the sterility of the environment where fabrication is taking place is crucial [10].

2.5 Silicon LOCs

The silicon is still used and the pioneering lab on chips was fabricated by using silicon. The disadvantages of using silicon mainly arise from the expensiveness of silicon, intransparency, lack of specialized microfabricaters and the need for sterility during fabrication. Silicon is inappropriate about high voltage requiring processes such as electrophoresis. The advantages of using silicon in production of lab on chips come due to the precision of silicon and the high variety of electrodes that can be used to interact with it [11].

3. LOCs and their application in different fields

Diazepam is a drug widely used in curing process of depression and anxiety. Apart from the health usage, diazepam is highly used as a recreational drug and while taken in high doses, it can cause debility, stomach’s harm, problems in eyes, unconsciousness and coma in rare occasions. LOC can be used about detecting high doses of it and it is highly beneficial for giving fast results. This LOC can be modified for detection of other drugs either harmful or health related [12]. Lab on chips for detection of pathogens, flow cytometry, clinical diagnosis, electrophoresis, DNA analysis, forensic science and chemical analysis of blood are among the state art technologies which are still under modification [13]. Recently lab on chips are being tested about single molecule studies. The high throughput ability of microfluidics is the key of a possible LOC development where measurements of picolitre or nanolitre scale can be possible. LOCs used for studying the electrophoretic movment of long molecules has been more efficient than Standard gel elctrophoresis. Similar researchers later combined a microfluidic device in a PCR therefore creating a capillary electrophoretic detection system. Afterwards the LOCs applying the principle of laminar flow and mixing were fabricated enabling the study of protein folding on the molecule level [14]. Research for development of LOCs related to fields, such as metabolomics, drug toxicity and stem cell differentation is highly active. Lab on chips which imitate vivo environments and allow interaction have been developed and they enable the precise control of any individual cell. Other LOCs have been modified in such a way that they allow the supplement and transfer of air, media and buffers. Also they have a good system for elimination of waste [15]. One of the latest lab on chips that is still not fully developed serves for separating and extracting the DNA belonging to the fetus. The principle they use is the usage of markers, such as free nucleic acids found in maternal plasma that belong to the foetal DNA. These LOCs can be used for detection of illnesses, such as Down Syndrome, haemolytic disease and sex determination. In the experimental uses this LOC has been shown to be more beneficial than the conventional

methods which are more prior to the DNA contamination. The glass material is being used for their fabrication [16]. The glass lab on chips are useful in rapid distinguishing between single and double DNA. The benefit of using glass is its sustainability. Also LOCs which analyse the single nucleotide polymorphisms have been developed apart from other LOCs which are used for genetic detections. Those LOCs are characterized by fast analysis time, real time detective ability and its minute size [17]. Lab on chips are still under development and the aim is to fabricate systems able to distinguish between the tumour cells and the healthy cells of a specific environment. The conventional methods used about isolation of the tumour cells from other cells are microscopic and molecular methods. The technology still being under development aims the combination of isolating and detecting ability. The minute size and the complexity of mixing in LOCs lead to problems, such as lack of efficiency in detecting the rare tumour cells. Some successful prototypes have been designed about studying the efficiency of chemotherapeutic agents used in treatment of cancer. A milestone has been achieved during those studies and a chip for measuring the variety of tumour responses toward different stress signals. It is advantagious to the conventional methods as it is lower in cost and faster [18]. Several experimental studies have been conducted about usage of LOCs in stem cells field and recently some achievements have occurred, such as identification of stem cells, real time studies of differentition and expansion of those cells. The state art technology related to the stem cells technology is the opportunity LOCs provide the researchers with: precise regulation of the conditions where cell culture is growing and the monitoring of several parameters at once [19]. LOCs used in embryo research are one of the most promising state art technologies of the microfluidic systems. Ethical issues related to the embryo research and problems caused by the difficulty of the process have been obstacles, but in the near future, the integration of an embedded electronic interface will support the development of a full automated embryo analysis system [20].

4. Advantages and Disadvantages of LOC

Development of lab on a chip has been a milestone due to various advantages it has, such as small surface to ratio and the large heat capacity [21]. Lab on a chip used for PCR has revolutionized the molecular biology as the process of amplification has become faster and faster genome sequencing is possible due to DNA array it can create [22]. The LOC’s usage in cell biology has enabled the control of various cells per unit of time and also it can be programmed to control a single cell due to its ability to detect and sort out [23]. LOC technology is a new opportunity about the unification of all proteomics steps, such as extraction, separation from the agglutination where it is found, electrophoresis analysis, mass spectroscopy and crystallography of protein [24]. LOCs are more economic than other methods of diagnostication and there are considered more sensitive to other methods. LOC saves time therefore making it a crucial technology for this period of time where extremely rapid solutions are needed. LOC is also very important as it enables the users to make real time monitoring. LOC is an innovative and sustainable solution as it is not voluminous and does not create a large quantity of waste [25]. LOC technology has some disadvantages due to the difficulty to the hard work and the specialized professionals about building lab on chips. As most of lab on chips are still on research they are not sufficient about a widespread use [26]. Properties of LOC such as its dependency on the capillary forces, chemical forces between the elements and the roghness of LOC’s surface convert the LOC create challenging problems that could be easily avoided if the conventional methods were used [27].

5. Latest Research on LOCs

Several interesting lab on the chips are still being under development and they are going to be very helpful in the respective field they are being manufactured for. One LOC related to the detection of the premature pregnancies, which is a risk factor for both female and baby’s life [28]. This year a lab on chip device which will be used for soil nutrient measurments is being developed. The scientists working to develop it have used the principle of capillary electrophoresis which is a good method about analyzing the ions in a fast and easy way. This will eliminate the usage of highly expensive tools which are normally used for analyzing the soil. The biggest challenges they have to overcome is the variability being caused by the chip differences, varying ionic strength and viscosity. This problem can be eliminated by using the bromide as an internal standard and during the soil measurements it is being dissipated from the chloride that is found everywhere in the soil by the usage of polyvinylpyrrolidone which acts as additive electrolyte. According to me, this LOC needs more studies with different control groups and more analysis are needed to adapt this method for real studies [29]. A lab on chip where different functions, such as Raman measurements and spectroscopic measurements are intended to be integrated is still under research. The LOC’s principle is the usage of an optical-fluidic jet waveguide. The detection scheme is based on the internal reflection of the liquid jet that has a radius approximately 75 µm. This is an innovative method and it is going to lead to a higher efficiency in exciting and collecting the signal. Its main advantage is that due to this technology it is going to avoid the sample container’s background and therefore minimize the errors in measurement. The device requires a small volume that is a good solution to the older measuring devices. There are still problems with this lab on chip as while comparing the conclusions it can be easily seen that there is a limit in practical detection those scientists have done. After some modifications, they achieved similar results to the ones calculated by the conventional methods. This means a new opportunity is arising for fabrication of better lab on chips that will change once and forever the Raman measurements and the mass spectroscopy methods, which are expensive and require high labor skills [30]. Optical sensors for detection and measurement of the glucose in saliva are under development. Nowadays the diabetes tests are based on using biosensors, where a sample of blood taken from the person’s finger is placed and the glucose level is measured. A lab on a chip method able to measure the glucose by analyzing the saliva in the patient’s mouth would solve the problem of finger pricking which can be apainful process for the patient. The detection and measurement principle of these LOCs will be based on the electromechanical and optical technology. Processes such as pretreatment of the saliva, mixing in the chip and measurement have to be unified in the LOC. The saliva which is going to be measured for the glucose level and glucose oxidase which is needed for the oxidation of the glucose will be placed on the inner channels of the chip. H2O2 which is going to be produced due to the reaction between glucose and glucose oxidase during the pretreatment proces of the glucose. In a third microchannel of the lab on a chip, a colorizing agent about establishing the mixing process. The measurement part is going to measure the absorbance value of the colorized mixture formed in the preceding step. Absorbance of the value 630 nanometers is considered to be the limiting value where the values lower than it represent a high glucose concentration in the saliva. In my opinion this method is not sufficient yet for use by diabetic persons as it does not provide certain quantitative precision which provide reliability to those types of measurements and some reliable health tests are needed to measure its efficiency in the measurement. In the near future, I expect that this LOC will be one of the most commercial LOCs. The high number of people who suffer from diabetes and the fact that this LOC will provide them with an opportunity to keep in real time check of their glucose level (therefore inhibiting any unpleasant scenario) are the reasons my opinion is based upon [31]. One of the most active research areas related to the LOCs is the diagnosis

and the manage of HIV infections. The approximate number of the people who suffer from HIV is 40 million and the number of sick people who are able to afford the anti-retroviral treatment is 1.3 million. Due to the statistics, only 10% of people infected with HIV have been diagnosed in an early phase of the virus. The flow cytometry is the traditional method to measure the CD4+ T lymphocytes which serve as a diagnostic marker for HIV. The disadvantages of this method arise due to the fact that this method is complicated, not affordable from every country, expensive and requires specialized people to perform the measurement. The LOC devices which are still under study will provide a solution to the problems caused by the traditional method [32]. Another LOC under development is the one which is going to be used for the characterization of polen tube on the Arabidopsis thaliana. The plant on a chip is a new concept which arose after this study. Practically, the pollen tissues and the ovules will be incubated and studied on the lab on the chip [33]. Lately scientist are aiming to generate self sustainable lab on chips. Those devices will be able to generate their own energy and therefore be a solution for the limited resources and it will be an efficeint solution for the remote regions. This future device is going to be known as miniaturized biological solar cells (Micro BSC-s). Their energy will be generated from the microbial photosynthetic and the daily respiratory activities. This technology does not exist stil as a practical application, because it has a low power. The experiments conducted about this technology where PDMS membrane is used, have demonstrated a biofilm production and electron transfer. The device was able to generate a power of approximately 18.6µW during days and 11.4µW·cm during nights. This experiment was repeated for 20 days, but still some other experiments related to the modification of this device are conducted about increasing the self power generation of those LOCs that would be a great opportunity for making LOCs affordable to all [34]. The unification of smart mobile devices LOCs and smart mobile devices is one the most challenging and most innovative technologies of the time. This field offers a great opportunity for personalized diagnostics and wide range affordable health check. Several problems associated to this field make it yet unready for consumer’s use, such as: the complexity of the lab on chips equipments and the integration problem. One of the applications it is being used on is the dopamine’s detection. The design of the lab on the chip device is planned to be dependant on a mobile software application that is coupled to a modular hardware accessory [35].

5.1 Lab in a Drop

Lab in a drop is going to be the future of lab on the chips, where the entire workflow of the diagnostic laboratory is going to be integrated within a single fluid drop. This new technology is stil under research and the detection principle will be based on the bioassay steps, such as: preparation of the sample, isothermal amplification of the target and detection of the amplicons’s readout [36].

5.2 Organ on Chip

Organ on chips are 3D cell culture models that aim to mimic the overall living organs, also including the biochemical reactions occuring in the living organism, mechanical properties of the cells and the various biological activity occurring on each organ. Theoretically, they are built with similar principles with the lab on chips devices. They are more innovative compared to the lab on chips and other methods of diagnostication, because the 3D model it is supported by is more flexible than the 2D models. This flexibility is supported by the changes caused by extracellular matrix and the cell-cell interations. Successful organ on chips, such as lung on chip, heart on chip, kidney on chip and artery on chip have been designed and several studies are being conducted for fabrication of other organs on chips. The main obstacles this

method faces to are the difficulty of those systems to mimic the complex interactions between different tissues, existence of temporal gradients caused by chemicals in vivo which affects the organs ‘s activity and the microenvironments created in different range of temperatures inside an organism [37].

5.3 Organism on a Chip

Organism on a chip is going to be a revolution for both organ on a chip and lab ona chip technology. In theory, the microfluidic elements will be implemented on various substrates consisting of different circuits through the process of direct microfabrication [38].

6. FUTURE PERSPECTIVE

LOC field is still under research and nowadays different worldwide are broadening and adapting this technology about using it in every possible field. The LOC technology is a promising field and in the near future the number of operations handled on the same chip are going to increase. Parallelization inside the same microfluidic channel is going to be the milestone of modern lab on chips. In the future the lab on chips can be easily sued even by unspecialized people and everyone will be able to perform several functions, such as cholesterol testing, glucose testing and similar crucial detections using a smartphone-LOC. The post classical medicine period will lead to a new form of medicine where real time complete diagnosis of a person will be possible. I believe that the doctors in the future will be needed only for the treatment process applications and the diagnosis process will be more personalized. Lab on a chip is a technology that will provide solution for low survival rates of several patients, reduce the antibiotic resistance and improve the treatment process. Due to this fascinating technology. I believe that in the future healthcare will not only be a luxury afforded only by the ones who can, but a right for everyone. The lab on chips development is a good opportunity due to the advance the persoanlized medicine has nowadays. Several limitations such as poor infrastructure, poor health data storage, recycling problems, limited resources about LOCs have to be overcome. In the same time several ethical issues, such as the unconsciousness usage about causing harm to another person or the loss of personal health data will arise as well. However, apart from the fact that LOC’s global distribution seems an utopian dream, the history is what has shown that the highest innovative discoveries always look impossible in their beginning. In my opinion the industry of smartphones where a lab on chip is going to be integrated will become part of our life sooner than we think. Recently, the development of blockchain technology [39] and the opportunity it offers for freely sharing and manipulating data. A system similar to the NCBI where all the genome data is protected would solve the software problem. On the other hand the personalized health websites, the drone technology for drugs transport and the policy makers would have to interact for regulation of such a huge system. I think that the self powering lab on chips devices would be a good solution about making those smartphone-lab on chips affordable for everyone.

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