Nanomedicine 1 - Bioanalysis Exam: Tested Questions and Answers, Exams of Biology

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What is the general principle of an affinity protein sensor? - CORRECT ANSWERS The general principle of an affinity protein sensor involves the use of a specific protein or peptide that can recognize and bind to a target protein of interest. This binding interaction between the affinity protein and the target protein leads to a measurable signal that can be used to detect and quantify the presence of the target protein. The affinity protein acts as a recognition element in the sensor, allowing for selective detection and analysis of the target protein in complex biological samples. In the case of affinity (non-catalytic) biosensor, the analyte is bound to the receptor irreversibly, and during the interaction no new biochemical reaction product is formed. This type of sensor comprises antibodies, cell receptors, and nucleic acids as the target for detection. Briefly mention possible transduction methods for protein biosensors. - CORRECT ANSWERS There are several possible transduction methods for protein biosensors. Some of the commonly used methods include: Optical methods: These involve the use of optical signals such as fluorescence, absorbance, or surface plasmon resonance (SPR) to detect and quantify the binding events between the affinity protein and the target protein. Electrochemical methods: These methods rely on the measurement of changes in electrical properties, such as current or potential, resulting from the binding interaction between the affinity protein and the target protein. Mechanical (Mass-based methods): These methods utilize the change in mass or resonance frequency caused by the binding event, which can be detected using techniques such as quartz crystal microbalance (QCM) or surface acoustic wave (SAW) sensors. What are the major difficulties in protein biosensor technology? - CORRECT ANSWERS 1. Selectivity and specificity

2. Stability and reproducibility
3. Sensitivity
4. Regeneration and reusability

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5. Sample matrix interference
6. Integration and miniaturization
7. Validation and standardization What are the basic/general elements (or components) of all point-of-care/Lab-on-a-chip biosensors? Include a list of five sensing principles. - CORRECT ANSWERS an analyte, bioreceptor, transducer, electronics, and display.
8. Analyte: A substance of interest whose constituents are being identified or detected (e.g., glucose, ammonia, alcohol, and lactose).
9. Bioreceptor: A biomolecule (molecule) or a biological element that can recognize the target substrate (i.e., an analyte) is known as bioreceptor (e.g., enzymes, cells, aptamers, deoxyribonucleic acid (DNA or RNA), and antibodies). The process of signal production (in the form of light, heat, pH, charge or mass change, plant or animal tissue, and microbial products) during the interaction between bioreceptor and analyte is called biorecognition.
10. Transducer: A device that transforms energy from one form to another. The transducer is a key element in a biosensor. It converts the biorecognition event into a measurable signal (electrical) that connects with the quantity or in the presence of a chemical or biological target. This process of energy conversion is known as signalization. Transducers generate either optical or electrical signals proportional to the number of analyte-bioreceptor interactions. According to the operating principle, transducers are broadly categorized as electrochemical, optical, thermal, electronic, and gravimetric transducers
11. Electronics: The transduced signal is processed and prepared for the display. The electrical signals obtained from the transducer are amplified and converted into digital form. The processed signals are quantified by the display unit.
12. Display: The display unit is composed of a user interpretation system, such as a computer or a printer that generates the output so that the corresponding response can be readable and understandable by the user. Depending on the end-user prerequisite, the output can be in the form of a numerical, graphical, or tabular value, or a figure.

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condensation of the ethoxysilane groups present in APTES, leading to the attachment of the amino group (-NH2) to the surface. Rinsing and Drying: After the silanization process, the surface is thoroughly rinsed with a suitable solvent to rem Briefly describe three methods for probing if you successfully have deposited biomolecules at the correct places. - CORRECT ANSWERS Fluorescence-based Imaging: Fluorescence-based imaging involves labeling the biomolecules of interest with fluorescent probes or dyes. If the biomolecules are successfully deposited at the desired locations, their fluorescence can be visualized and detected using fluorescence microscopy or imaging systems. The presence of fluorescence signals confirms the correct deposition of biomolecules at the desired spots. Atomic Force Microscopy (AFM): Atomic Force Microscopy is a powerful technique that can probe the topography and physical properties of surfaces at the nanoscale. By scanning the surface with a sharp tip, AFM can provide high-resolution images of the deposited biomolecules. Successful deposition can be confirmed by observing the presence of biomolecules at the expected locations with specific features and patterns. Surface Plasmon Resonance (SPR): Surface Plasmon Resonance is a label-free technique that can detect biomolecular interactions in real-time. By immobilizing a receptor or ligand on a surface, changes in the refractive index resulting from the binding of biomolecules can be measured. Successful deposition is indicated by the detection of specific binding events at the desired locations, confirming the presence of the deposited biomolecules.

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To chemically conjugate an antibody to a silicon/glass surface there are many options, please describe one method to achieve this. - CORRECT ANSWERS One method to chemically conjugate an antibody to a silicon/glass surface is through the use of silane coupling agents = silanization. Surface preparation: Thoroughly clean the silicon/glass surface to remove contaminants. Silane coupling agent selection: Choose a suitable silane coupling agent with functional groups that can react with both the surface and the antibody. Silanization process: Prepare a solution of the silane coupling agent and immerse or apply it onto the surface. Allow the surface to react with the silane to form covalent bonds. Antibody conjugation: Prepare a solution of the antibody and incubate the silanized surface with the antibody solution. Control incubation time and conditions for optimal binding. Wash the surface to remove unbound or non-specifically adsorbed antibodies. Optional: Block the surface with blocking agents like BSA or casein to minimize non-specific interactions and enhance stability. Since a complete absorption of a sample analyte to a biosensor surface will not be feasible as measuring principle due to varying concentrations, what other principles must be employed to get accurate quantitative measurements. - CORRECT ANSWERS - One crucial principle is signal transduction, where the interaction between the analyte and the sensing element is converted into a measurable signal. Biosensors utilize various transduction methods such as optical, electrochemical, piezoelectric, or thermal-based techniques. The generated signal is proportional to the analyte concentration, allowing for quantitative measurements.

• Calibration curves play a vital role in biosensors. By measuring the signal response at known analyte concentrations and plotting them, a calibration curve is generated. This curve establishes the relationship between the measured signal and the analyte concentration, enabling the determination of unknown sample concentrations.
• Standardization and reference materials are employed to ensure accuracy and comparability. Standard samples with known analyte concentrations are used to calibrate

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• Additionally, creating a calibration curve with multiple standards covering a wide range of concentrations helps compensate for the limited dynamic range of the biosensor, enabling more accurate measurements across different analyte concentrations. You want to look at the communication between two cell types and need to organize the cells in regular patterns. You decide to use surface functionalization on glass. Briefly describe three methods for determining if the surface has been functionalized. - CORRECT ANSWERS
• Contact Angle Measurement: Contact angle measurement is a common technique used to assess the wettability of a surface. By measuring the contact angle formed between a liquid droplet and the functionalized surface, one can determine the hydrophilicity or hydrophobicity of the surface. If the surface has been successfully functionalized, it may exhibit a different contact angle compared to an untreated or non-functionalized surface.
• X-ray Photoelectron Spectroscopy (XPS): XPS is a surface-sensitive technique used to analyze the elemental composition and chemical states of a material. By subjecting the functionalized surface to X-ray irradiation, the emitted photoelectrons can be detected and analyzed to identify the presence of specific functional groups or chemical moieties that were introduced during the surface functionalization process.
• Fluorescence Imaging: Fluorescent labeling or tagging of the functional groups can be employed to visualize and confirm the successful surface functionalization. This can be achieved by attaching fluorescent molecules or dyes to the functional groups or using specific antibodies or probes that selectively bind to the functionalized molecules. Fluorescence imaging techniques, such as confocal microscopy or fluorescence microscopy, can then be used to observe and verify the presence of the functionalized surface through the detection of fluorescence signals. Outline one process for patterning one protein on glass - CORRECT ANSWERS Microcontact printing = a technique used for patterning proteins or other biomolecules onto a substrate, such as glass. It involves the transfer of molecules from an elastomeric stamp to the substrate surface through a process of direct contact and selective adsorption.

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• Stamp preparation: A stamp is fabricated using a polydimethylsiloxane (PDMS) elastomer, which is typically molded from a master template. The master template is created by photolithography or other patterning techniques.
• Ink deposition: The stamp is then coated with a protein-containing ink or solution. The ink can be prepared by incorporating the desired protein or biomolecule into an appropriate solvent or buffer.
• Contact with substrate: The stamp is brought into contact with the glass substrate, allowing the protein to transfer from the stamp to the substrate surface. The contact between the stamp and substrate is typically facilitated by applying a small pressure or using a controlled mechanical force.
• Protein adsorption: The protein molecules selectively adsorb onto the substrate in the regions where the stamp comes into contact, resulting in the formation of a patterned protein layer.
• Stamp removal: The stamp is carefully peeled off from the substrate, leaving behind the patterned protein layer on the glass surface.
• Microcontact printing offers several advantages for protein patterning. It allows for precise control over the spatial arrangement of proteins, enabling the creation of complex patterns and structures. It is a relatively simple and cost-effective technique that does not require sophisticated equipment. However, it is important to note that the success of microcontact printing depends on factors such as stamp quality, ink formulation, and surface chemistry of the substrate, which need to be carefully optimized for each spec You want to pair cells 1:1 at a large scale for a cell:cell interaction experiment. Sketch a microfluidic device - and outline the operational instructions for it - that can be useful for achieving efficient cell pairing. - CORRECT ANSWERS -> Cell traps Three-step cell loading protocol. (a) Cells are first loaded "up" towards the smaller backside capture cup. (b) The direction of the flow is reversed, and the cells are transferred "down" into the larger frontside capture cup 2 rows below; scale bar, 50 μm. (c) The second cell type is loaded in from the top, and cells are captured in front of the first cell type.

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List advantages and disadvantages of using PDMS - poly(dimethylsiloxane) - in microfluidic cell biological applications. - CORRECT ANSWERS Advantages:

• Excellent sealing, between glass and PDMS
• Flexible and soft material
• Easy for connecting tubing adapters
• Transparent material: great for microscopic observation
• Allow multi-layer process toward 3D networks
• Biocompatible: suitable for cell culture and interaction studies Disadvantages:
• Absorption: has a tendency to absorb small molecules -> can lead to loss of concentration or altered experimental conditions.
• Hydrophobic surface properties: can lead to issues such as air bubble trapping, non- uniform flow, and nonspecific adsorption of biomolecules.
• Limited chemical compatibility: PDMS is not compatible with certain organic solvents, acids, and bases, as it can swell or degrade in their presence.
• Poor long-term stability: PDMS is prone to physical and chemical degradation over time, especially under harsh conditions such as high temperatures or prolonged exposure to UV radiation.
• Difficult bonding to other materials: PDMS does not readily bond to other materials, which can make integration with additional components or systems challenging. Describe the working principle of 2-photon polymerization for 3D printing - CORRECT ANSWERS - = Femtosecond (Fs) laser pulses that directly write the pattern into the volume of the photo curable resin. In 2PP, two photons are being absorbed simultaneously by the photo initiator enabling them to act as one photon to start polymerization. -> allows the

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laser to direct record or writes any desired polymeric 3D pattern into the volume of photosensitive materials = Two-photon polymerization (2PP) is a 3D printing technique that relies on non-linear absorption of photons in a photosensitive resin. A tightly focused ultrafast laser beam with a near-infrared wavelength is used to polymerize the resin. The high laser intensity at the focal point leads to the simultaneous absorption of two photons by a photo initiator, initiating a photochemical reaction. This localized polymerization allows for the fabrication of complex 3D structures with high resolution. By scanning the laser beam layer by layer, the desired object is built up. The precise control of laser parameters enables the creation of structures with varying feature sizes and shapes. The use of a near-infrared laser allows for deep penetration into the resin, enabling the fabrication of intricate internal structures within the printed object. Overall, 2PP offers a precise and versatile method for creating intricate 3D objects with fine detail and complex geometries. Please outline the process of making a microfluidic chip in PDMS - CORRECT ANSWERS 1. Design the microfluidic channel layout using CAD software.

2. Prepare the PDMS mixture by thoroughly mixing the base polymer and curing agent.
3. Pour the PDMS mixture onto a master mold and remove air bubbles through degassing.
4. Cure the PDMS by heating it to initiate the cross-linking reaction.
5. Peel off the PDMS from the mold to obtain the replica of the microfluidic channels.
6. Bond the PDMS chip to other surfaces or substrates, such as glass or additional PDMS layers.
7. Apply surface treatments, if needed, to modify the surface properties.
8. Integrate the microfluidic chip with other components and create inlet and outlet ports.
9. The microfluidic chip is now ready for use in fluidic experiments.

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What are the basic structures that are found in tissues? - CORRECT ANSWERS Tissue architecture:

• Microniches: interactions with ECM
• Functional units
• Compartmentalization
• Vascularization
• Interconnection (other tissues, air, food) EVT. Cells, extracellular matrix (ECM), cell junctions, blood vessels, and nerve fibers. These structures work together to provide support, organization, communication, and specialized functions within tissues. What are the five strategies (cues and contents) necessary to build proper organs-on-chips in hydrogel biomaterials? - CORRECT ANSWERS 1. Biomimetic ECM: Mimic the composition and structure of the native extracellular matrix.

2. Cell-Cell Interactions: Promote cell clustering and communication by incorporating cell adhesion molecules and multiple cell types.
3. Mechanical Properties: Tailor the hydrogel's stiffness and elasticity to replicate the mechanical cues of native tissues.
4. Vascularization: Integrate vascular-like networks within the hydrogel to mimic blood vessel functionality.
5. Physiological Stimuli: Apply appropriate physiological stimuli, such as fluid flow, electrical stimulation, or mechanical forces, to replicate the dynamic environment of the organ. -These strategies ensure that the hydrogel-based organs-on-chips closely resemble the native tissue architecture and provide a suitable microenvironment for cellular behavior and organ functionality.

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Describe a way of creating true 3D structures in hydrogels. - CORRECT ANSWERS Inkjet bioprinting = utilizes a modified inkjet printer to deposit small droplets of bioink, which is a mixture of hydrogel and cells, onto a substrate in a precise and controlled manner. The droplets form successive layers that eventually build up into a 3D structure.

• -> The bioink is loaded into the printer cartridge, and the printer ejects the droplets onto the substrate using thermal or piezoelectric mechanisms. The printer follows a predetermined pattern based on a digital model or design, allowing for the precise positioning of the droplets.
• -> The bioink droplets contain living cells suspended in a hydrogel matrix. Once deposited, the hydrogel can undergo crosslinking or gelation to stabilize and solidify the structure. This can be achieved through various methods, such as chemical crosslinking agents, light- induced crosslinking, or temperature-induced gelation, depending on the specific hydrogel used.
• -> Inkjet bioprinting offers advantages such as high resolution, scalability, and the ability to handle multiple cell types or bioactive molecules. It has found applications in tissue engineering, regenerative medicine, drug screening, and other areas where the creation of precise and complex 3D structures in hydrogels is required. Outline three different approaches for micro structuring in hydrogels. - CORRECT ANSWERS Photolithography:
• a commonly used approach for micro structuring hydrogels. It involves creating a photomask with a specific pattern and exposing a photosensitive hydrogel to UV light through the mask. The exposed regions undergo a chemical reaction or crosslinking, while the unexposed regions remain unaltered. Subsequent rinsing removes the uncross linked portions, resulting in a microstructured hydrogel with precise features. 3D Bioprinting:
• enables the precise deposition of hydrogel materials layer by layer to create complex 3D structures. It involves preparing a bioink, containing the hydrogel and other components, and using a bioprinter to dispense the bioink according to a digital design. The bioink is often

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• Dissolvable network-based sacrificial molding = at first a 2D or 3D network of a negative mold is formed using an easily dissolvable gel or solid material. The pre-formed mesh or network is then encapsulated in a 3D hydrogel. Finally, the sacrificial mesh or network is dissolved or melted and flushed out from the gel matrix, leaving behind interconnected channels in the hydrogel structure. Vasculogenesis and angiogenesis-based techniques - micropatterning method: Micromolding = is a widely used method for micropatterning cell-laden hydrogels. It involves transferring the shape of a preformed master pattern onto a prepolymer solution through direct contact, leading to crosslinking of the prepolymer. PDMS is commonly used as the material for the master mold. This technique enables the design of constructs with spatially patterned endothelial cell (EC) tubular structures of various geometrical properties. It has been employed to promote capillary tubule formation by patterning ECs using long tubes of collagen hydrogel. The size of the resulting lumens can be controlled by adjusting the microgel size and the concentration of collagen. This approach holds promise for developing complex blood vessel-like structures, allowing for the study of the behavior of ECs during tubulogenesis initiation. Please outline a fabrications strategy to achieve a working muscle in miniature chip format. - CORRECT ANSWERS Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three- dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies.
• Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers.
• LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with successful development of diseased 3D myotubes displaying reduced contraction. The miniaturized

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myotube technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Please outline three approaches for creating blood vessel networks on a chip - CORRECT ANSWERS 1. Subtractive sacrificial methods

• Dissolvable network-based sacrificial molding = In the dissolvable-network-based sacrificial molding, at first a 2D or 3D network of a negative mold is formed using an easily dissolvable gel or solid material. The pre-formed mesh or network is then encapsulated in a 3D hydrogel. Finally, the sacrificial mesh or network is dissolved or melted and flushed out from the gel matrix, leaving behind interconnected channels in the hydrogel structure.

2. Direct ink writing (DIW) = a 3D printing technique that enables the creation of intricate structures, including blood vessel networks, on a chip. It involves the precise extrusion of a bioink, which is a combination of a hydrogel material and living cells, to fabricate the desired 3D architecture. The bioink is dispensed through a syringe-based extrusion system, allowing for controlled deposition of the material to create the blood vessel network. The key principle of DIW is the ability to directly write or deposit the bioink in a layer-by-layer fashion, enabling the fabrication of complex, multi-scale structures with high precision and resolution. This approach allows for the recreation of vascular networks in a controlled manner, providing a platform for studying blood flow, cell-cell interactions, and other aspects of vascular biology in a microscale environment.
3. Bonding of hydrogel layers (additive method) = a 3D network of interconnected channels is formed by layer-by-layer stacking and bonding of pre-formed planer hydrogel slabs. The slabs contain network of channels featured in 2D planes, pre-formed using micropatterning techniques such as photomask lithography or micromolding in such a way that when stacked together they result in a 3D network of channels. The adjacent layers are bonded together irreversibly using partial meltin