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 .
Keywords: organ on the chip, microfluidics, biosensors
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 .
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 . 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 . 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. 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 . The generation of an electrochemical biosensor was realized by coating a gold microelectrode with a self assembled monolayer by using 11-MUA  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 . 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 . 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 .
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 . 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 . 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 . 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 ) . 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 . 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 . 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
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 .
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 . 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 . 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 . 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.
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.
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
5. Inamdar, N. K. & Borenstein, J. T. Microfluidic cell culture models for tissue engineering. Curr. Opin. Biotechnol. 22, 681–689 (2011).
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.