Imaging and measurement Final Project

Final Project: Measurement Plan

Recombinant Piezoelectric Biomaterial Interface for Soft Robotic Muscle Tone Modulation

Construct: His₆-FLAG-TEV-NQEQVSPL-(GGGGS)₃-GRGDS-IKVAV-(GPP)₁₀ in pcDNA3.1(+) / HEK293T expression system

Question 1: What aspects of the project will be measured?

This project proposes the design, synthesis, and initial characterisation of a recombinant piezoelectric fusion protein intended as the bioactive interface layer of a soft robotic rehabilitation device. The protein is engineered to: (1) be correctly expressed in human cells, (2) fold into a structurally active conformation — particularly the collagen-like (GPP)₁₀ domain — and (3) generate measurable electrical output under mechanical compression. Together, these three properties constitute the scientific claim of the project.

To evaluate whether each of these claims is substantiated, measurements are organised across three categories:

• Category 1 — DNA-level measurements: Confirm that the plasmid construct is assembled correctly, the insert sequence matches the design, and the DNA is of sufficient purity for downstream experiments.
• Category 2 — Protein-level measurements: Confirm that the recombinant protein is expressed in HEK293T cells, is present at detectable yield, adopts the correct secondary structure (triple helix in the collagen domain), and that the bioactive motifs (RGD and IKVAV) are functionally active.
• Category 3 — Functional measurements: Confirm that the cast protein film generates voltage under mechanical deformation (the piezoelectric effect), is mechanically compatible with soft tissue, and is non-cytotoxic to muscle cells.

In total, 12 distinct measurements are performed, using 7 core analytical technologies.

Question 2: Description of all elements to be measured and how measurements will be performed

Category 1: DNA-level measurements

Measurement 1 — Plasmid insert sequence identity

What is measured: The exact nucleotide sequence of the full 282 bp expression cassette inserted into pcDNA3.1(+), including the Kozak sequence, His₆ tag, FLAG tag, TEV protease site, piezoelectric domain (NQEQVSPL), three GGGGS flexible linkers, RGD adhesion motif (GRGDS), IKVAV neural attachment motif, (GPP)₁₀ collagen-like domain, and stop codon. Every base must match the codon-optimised design.

How it is performed: To verify the correct insertion and sequence identity of the piezoelectric fusion protein cassette within the pcDNA3.1(+) expression vector, Sanger dye-terminator sequencing was performed on the final assembled plasmid. Following maxiprep purification (Qiagen HiSpeed Maxi Kit) of plasmid DNA propagated in E. coli DH5α, the concentration and purity of the DNA were confirmed by Nanodrop spectrophotometry, with acceptable samples yielding an A₂₆₀/A₂₈₀ ratio between 1.8 and 2.0 and a concentration of at least 100 ng/µL. Two sequencing reactions were submitted per sample: a forward read using the standard T7 promoter primer (5′-TAATACGACTCACTATA-3′), which anneals upstream of the multiple cloning site and reads into the insert from the CMV promoter end, and a reverse read using the BGH reverse primer (5′-TAGAAGGCACAGTCGAGG-3′), which anneals downstream of the insert and reads back through the 3′ end of the coding sequence. Together, these two reads provide overlapping coverage of the full 282 bp insert. Sequencing was performed by an external provider using capillary electrophoresis of fluorescently labelled chain-termination fragments. The resulting chromatogram trace files (.ab1 format) were uploaded to Benchling and aligned to the expected construct sequence using the built-in pairwise alignment tool.

Technologies used: Sanger sequencing, capillary electrophoresis, Benchling sequence alignment, colony PCR (pre-screening)

Pass criterion: 100% base-call agreement with the expected sequence across the full 282 bp insert. Zero frameshifts, zero unexpected stop codons, all nine annotated domains present in the correct order and reading frame.

Measurement 2 — Correct plasmid size and insert presence (analytical restriction digest)

What is measured: The size of DNA fragments produced after cutting the assembled plasmid with HindIII and XhoI restriction enzymes. This confirms that the insert was ligated into the backbone at the correct sites and is the expected size.

How it is performed: Following sequence verification, 500 ng of maxiprep plasmid was digested in a 20 µL reaction containing 1 µL each of HindIII-HF and XhoI (New England Biolabs), 2 µL CutSmart buffer, and nuclease-free water. The reaction was incubated at 37°C for 60 minutes and heat-inactivated at 65°C for 20 minutes. The digested products were resolved on a 2% agarose gel prepared in 1× TAE buffer containing GelRed nucleic acid stain (1:10,000 dilution). A 1 kb Plus DNA ladder (NEB) was loaded alongside the samples. Electrophoresis was performed at 100V for 45 minutes. The gel was imaged under UV illumination using a gel documentation system. Band positions were compared to the expected fragment sizes calculated from the plasmid sequence.

Technologies used: Restriction enzyme digestion, agarose gel electrophoresis, UV gel imaging

Pass criterion: Two bands visible — approximately 282 bp (insert fragment) and 5,428 bp (linearised backbone). Absence of the smaller band indicates failed cloning or incorrect insertion orientation.

Measurement 3 — DNA purity and concentration (pre-transfection quality control)

What is measured: Concentration of purified plasmid DNA in ng/µL and purity ratios A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀, which reflect contamination by protein, phenol, or chaotropic salts respectively. This confirms the DNA preparation is suitable for HEK293T transfection.

How it is performed: After maxiprep purification, the Nanodrop ND-1000 spectrophotometer (Thermo Fisher) was blanked with the elution buffer used in the final step of the purification protocol. A 1 µL aliquot of the plasmid preparation was loaded on the measurement pedestal. Absorbance readings at 230, 260, and 280 nm were recorded. Concentration was calculated from A₂₆₀ using the conversion factor for double-stranded DNA (50 ng·cm/µL). Samples were diluted to a working concentration of 1 µg/µL in TE buffer for transfection.

Technologies used: Nanodrop UV spectrophotometry

Pass criterion: A₂₆₀/A₂₈₀ ratio 1.8–2.0 (pure DNA). A₂₆₀/A₂₃₀ ≥ 1.8. Concentration ≥ 500 ng/µL. Samples outside these ranges were re-purified before use in cell transfection experiments.

Category 2: Protein-level measurements

Measurement 4 — Protein expression detection (western blot)

What is measured: Presence of the recombinant piezoelectric fusion protein in HEK293T cell lysate, detected by antibody recognition of the His₆ or FLAG epitope tag. The expected molecular weight of the full fusion protein including tags is approximately 13.6 kDa.

How it is performed: HEK293T cells were seeded at 2 × 10⁶ cells per well in 6-well plates and grown to 70–80% confluency in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were transiently transfected with 2 µg of the sequence-verified pcDNA3.1(+)-Piezo plasmid using Lipofectamine 3000 reagent (Thermo Fisher) according to the manufacturer’s protocol. An untransfected well was maintained as a negative control. At 48 hours post-transfection, cells were washed with ice-cold PBS and lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) supplemented with protease inhibitor cocktail. Protein concentration in the clarified lysate was determined by BCA assay (Pierce). Twenty micrograms of total protein per lane were resolved on a 15% SDS-polyacrylamide gel under denaturing conditions and transferred to a 0.2 µm PVDF membrane by wet transfer at 100V for 60 minutes. The membrane was blocked with 5% non-fat dried milk in TBST for 1 hour at room temperature, then incubated overnight at 4°C with either mouse anti-His (1:2000, Abcam ab18184) or mouse anti-FLAG (1:1000, Sigma F1804) primary antibody. After washing (3 × 10 min in TBST), the membrane was incubated with HRP-conjugated anti-mouse IgG secondary antibody (1:5000) for 1 hour at room temperature. Signal was detected by enhanced chemiluminescence (ECL) using a ChemiDoc imaging system.

Technologies used: Cell transfection (Lipofectamine 3000), SDS-PAGE, wet transfer, PVDF western blot, chemiluminescence imaging

Pass criterion: A single band at approximately 13.6 kDa in the transfected lane, absent in the untransfected control. Presence of additional bands at higher molecular weight may indicate incomplete denaturation of the collagen triple helix domain (which is SDS-resistant in some contexts) and should be noted.

Measurement 5 — Protein yield quantification after affinity purification

What is measured: Total mass (mg) of purified recombinant piezoelectric protein recovered per litre of HEK293T culture, following Ni-NTA affinity chromatography via the His₆ tag.

How it is performed: HEK293T cells transfected as described in Measurement 4 were scaled to T-175 flasks. At 48–72 hours post-transfection, cells were harvested, lysed in native lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) and the clarified lysate applied to a pre-equilibrated Ni-NTA agarose column (Qiagen). The column was washed with 20 column volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0) and the protein eluted in elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). Eluted fractions were dialysed into PBS to remove imidazole. Protein concentration was determined by BCA assay using a BSA standard curve (0–2000 µg/mL). Absorbance at 562 nm was read on a plate reader (Tecan Infinite M200) after 30 minutes incubation at 37°C with BCA reagent.

Technologies used: Ni-NTA affinity chromatography, BCA protein assay, plate reader spectrophotometry at 562 nm

Pass criterion: ≥ 0.5 mg/L culture (transient HEK293T). Lower yields are acceptable at proof-of-concept stage but would necessitate stable cell line generation or bioreactor scale-up for material production.

Measurement 6 — Exact protein molecular mass and post-translational modifications (mass spectrometry)

What is measured: The precise molecular mass of the purified protein to confirm identity and detect post-translational modifications — in particular hydroxyproline (+16 Da per modified residue) as evidence that the endogenous HEK293T prolyl-4-hydroxylase (P4H) has processed the (GPP)₁₀ collagen-like domain.

How it is performed: Purified protein (≥ 5 µg in low-salt PBS, ≤ 150 mM NaCl) was submitted to the institutional mass spectrometry core facility for analysis by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. For intact protein analysis, the sample was co-crystallised with α-cyano-4-hydroxycinnamic acid (CHCA) matrix. For peptide-level confirmation of domain composition and modification sites, a parallel aliquot was digested with sequencing-grade trypsin (1:50 enzyme:protein ratio, 37°C overnight) and analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on a Q Exactive Orbitrap instrument. Raw spectra were processed using MaxQuant and searched against the expected protein sequence. Hydroxyproline was included as a variable modification (+15.9949 Da on Pro residues).

Technologies used: MALDI-TOF mass spectrometry, tryptic digest, LC-MS/MS, MaxQuant database search

Pass criterion: Intact mass within ±0.5% of the theoretical mass (6,900 Da for the tag-cleaved insert; 13,600 Da with His₆-FLAG-TEV tags). Detection of +16 Da shifts on proline residues in the (GPP)₁₀ domain confirms hydroxyproline formation and successful P4H activity, validating the use of HEK293T as expression host.

Measurement 7 — Collagen domain triple helix formation (circular dichroism spectroscopy)

What is measured: The secondary structure of the purified fusion protein — specifically whether the (GPP)₁₀ domain adopts a polyproline type-II (PPII) triple helix, which is both the structural and functional prerequisite for piezoelectric activity in collagen-derived materials. The melting temperature (Tm) is also measured as an indicator of thermostability at physiological temperature.

How it is performed: Purified protein was dialysed into CD-compatible buffer (10 mM sodium phosphate, pH 7.4, no chloride ions which absorb in the far-UV) and diluted to 0.3–0.5 mg/mL. Circular dichroism spectra were recorded at 4°C on a Jasco J-815 spectropolarimeter using a 1 mm path-length quartz cuvette (Hellma). Wavelength scans were performed from 190 to 260 nm at 1 nm intervals, 1 nm bandwidth, 1 second response time, and three accumulations averaged per spectrum. The buffer baseline was subtracted. Data were expressed as mean residue ellipticity (MRE, deg·cm²·dmol⁻¹). A collagen triple helix produces a characteristic signature: a positive peak near 225 nm and a negative peak near 200 nm. To determine thermal stability, a thermal denaturation scan was performed by monitoring ellipticity at 225 nm while ramping temperature from 4°C to 70°C at 1°C/min. Tm was calculated as the inflection point of the sigmoidal melting curve using a Boltzmann fit.

Technologies used: Circular dichroism spectroscopy, thermal denaturation, Boltzmann curve fitting

Pass criterion: A positive CD signal at approximately 225 nm confirms PPII triple helix formation. Tm ≥ 30°C confirms the domain is stable approaching physiological temperature. A Tm ≥ 37°C would be required for any future in vivo application. Absence of the 225 nm signal indicates the collagen domain is unfolded — likely due to insufficient GPP repeat length or absent hydroxyproline — and the construct would require redesign.

Measurement 8 — Functional activity of RGD and IKVAV bioactive motifs (cell adhesion assay)

What is measured: The ability of the purified fusion protein — specifically the GRGDS and IKVAV domains — to promote adhesion of C2C12 skeletal muscle myoblasts to a coated surface relative to an uncoated control and a fibronectin positive control.

How it is performed: Flat-bottomed 96-well plates were coated overnight at 4°C with 100 µL per well of the purified piezoelectric protein at 10 µg/mL in PBS. Uncoated wells (PBS only) served as negative controls; fibronectin-coated wells (10 µg/mL) served as positive controls. Wells were blocked with 1% BSA in PBS for 1 hour at room temperature to prevent non-specific adhesion, then washed three times with PBS. C2C12 myoblasts (ATCC CRL-1772) were detached with trypsin-EDTA, resuspended in serum-free DMEM, and 10,000 cells per well seeded in 100 µL. Plates were incubated at 37°C with 5% CO₂ for 2 hours. Non-adherent cells were removed by three gentle washes with PBS. Adherent cells were fixed with 4% paraformaldehyde for 10 minutes, stained with 0.1% crystal violet in 25% methanol for 20 minutes, washed five times with distilled water, and air-dried. Crystal violet was solubilised by addition of 10% acetic acid (100 µL/well) with shaking for 10 minutes. Absorbance at 590 nm was measured on a plate reader. All conditions were performed in triplicate (n = 3 independent experiments).

Technologies used: Cell adhesion assay, crystal violet staining, plate reader at 590 nm, fluorescence microscopy (optional confirmation)

Pass criterion: ≥ 2× A₅₉₀ signal on protein-coated wells relative to uncoated control (p < 0.05 by one-way ANOVA with Tukey post-hoc). Adhesion comparable to fibronectin positive control would constitute a strong result. Failure to exceed uncoated control suggests the RGD/IKVAV domains are sterically occluded in the folded protein conformation.

Category 3: Functional measurements

Measurement 9 — Piezoelectric voltage output under mechanical compression

What is measured: The voltage (mV) and piezoelectric coefficient (d₃₃, expressed in pC/N) generated by the cast protein film when subjected to controlled, reproducible compressive mechanical stress. This is the primary functional readout of the entire project — the measurement that directly tests whether the designed material does what it is intended to do.

How it is performed: Purified protein was cast as a film on gold-coated ITO glass substrates at a concentration of 20 µg/cm² by slow evaporation at room temperature in a humidity-controlled environment. Films were air-dried for 24 hours and their thickness measured by profilometry (expected: 50–200 nm). For macroscale electrical characterisation, a top gold electrode was deposited by sputter coating. Films were connected to a high-impedance electrometer (Keithley 6517B, input impedance > 200 TΩ) to prevent charge dissipation. A sinusoidal compressive force (1 Hz, amplitude 1–10 N) was applied perpendicular to the film surface using a dynamic mechanical analyser (TA Instruments DMA Q800) fitted with a flat compression clamp. The voltage output waveform was recorded simultaneously on a digital oscilloscope (Tektronix TBS1052B) at a sampling rate of 10 kHz. The piezoelectric coefficient d₃₃ was calculated as d₃₃ = Q/F, where Q is the generated charge (integrated current) and F is the applied force. For nanoscale domain mapping, piezoresponse force microscopy (PFM) was performed on a Bruker Dimension Icon AFM in contact mode using a conductive Pt/Ir-coated cantilever, applying an AC bias of 2V at 20 kHz to map the piezoelectric response across the film surface.

Technologies used: Dynamic mechanical analyser, high-impedance electrometer, digital oscilloscope, piezoresponse force microscopy (PFM), gold sputter coating, film profilometry

Pass criterion: A measurable, reproducible voltage output in phase with the applied mechanical force, with d₃₃ ≥ 1 pC/N. This threshold is benchmarked against natural collagen (0.7–2 pC/N) and represents the minimum signal relevant for neuromuscular stimulation. Even a sub-threshold signal at this stage constitutes a positive scientific result, as no prior study has measured piezoelectric output from a sequence-designed recombinant protein of this composition.

Measurement 10 — Mechanical stiffness of the protein film (Young’s modulus)

What is measured: The elastic modulus (Young’s modulus, kPa) of the hydrated protein film, which determines whether the material is mechanically compatible with direct skin and muscle tissue contact in a wearable device. A material that is too stiff creates compliance mismatch; a material that is too soft lacks structural integrity.

How it is performed: Protein films were cast on glass substrates as described above and hydrated in PBS at 37°C for 1 hour prior to measurement to replicate physiological conditions. Nanoindentation was performed using atomic force microscopy (AFM, Bruker BioScope Catalyst) in force-volume mode. A pyramidal silicon nitride cantilever (spring constant k = 0.03 N/m, tip radius approximately 20 nm, Bruker MLCT) was calibrated against a glass slide. Force-indentation curves were collected at 25 locations distributed across the film surface at an indentation depth of 200–500 nm. Young’s modulus was calculated from each curve by fitting to the Hertz contact model for a pyramidal indenter using NanoScope Analysis software. For bulk mechanical characterisation, dog-bone shaped film specimens were punched with a custom die (gauge length 5 mm, width 2 mm) and tested on a micro-tensile tester (Instron 5943) at a crosshead displacement rate of 1 mm/min. Stress-strain curves were recorded and Young’s modulus extracted from the linear elastic region (strain 0–5%).

Technologies used: AFM nanoindentation, Hertz contact model fitting, uniaxial tensile testing

Pass criterion: Young’s modulus 1–100 kPa (skeletal muscle stiffness: 8–17 kPa; soft tissue wearable contact range: 1–100 kPa). Films with modulus > 500 kPa would be considered mechanically mismatched for skin-contact applications and would require reformulation as a composite hydrogel.

Measurement 11 — Cytotoxicity of the protein film (Live/Dead fluorescence assay)

What is measured: The percentage of live C2C12 skeletal muscle myoblasts after 72 hours of direct culture on the protein film surface, compared to a tissue-culture polystyrene control and a known toxic positive control (0.1% Triton X-100). This establishes that the material is not cytotoxic — a prerequisite for any future in vivo or clinical application.

How it is performed: Protein films were cast directly into the wells of a 24-well tissue culture plate (10 µg/cm²) and allowed to dry. Films were sterilised by exposure to UV light (254 nm, 30 minutes) in a biosafety cabinet. Wells were rehydrated with PBS, blocked with 1% BSA, and then 50,000 C2C12 cells per well were seeded in complete DMEM. After 72 hours of culture at 37°C with 5% CO₂, media was aspirated and wells washed gently twice with PBS. The Live/Dead staining solution was prepared by diluting Calcein AM to 2 µM and ethidium homodimer-1 (EthD-1) to 4 µM in PBS. One hundred microlitres of staining solution was added per well and incubated at room temperature for 20 minutes protected from light. Cells were imaged immediately on an inverted fluorescence microscope (Zeiss Axio Observer) using FITC filter (Calcein AM, live cells: green fluorescence, excitation 495 nm, emission 515 nm) and TRITC filter (EthD-1, dead cells: red fluorescence, excitation 528 nm, emission 617 nm). Five randomly selected fields per well were imaged at 10× magnification. Live and dead cells were counted using the Cell Counter plugin in Fiji/ImageJ. Percentage viability was calculated as (live cells / total cells) × 100.

Technologies used: Fluorescence microscopy, Calcein AM / EthD-1 Live/Dead assay, Fiji/ImageJ cell counting

Pass criterion: ≥ 80% cell viability on the protein film compared to uncoated tissue-culture plastic control (baseline viability expected ≥ 95%). ISO 10993-5 standard for in vitro cytotoxicity: less than 30% reduction in viability relative to control = non-cytotoxic classification. A result below 70% viability would require investigation of residual imidazole contamination from purification or osmolarity effects from film preparation.

Measurement 12 — Protein thermal stability (differential scanning fluorimetry)

What is measured: The melting temperature (Tm, °C) of the full fusion protein — the temperature at which 50% of the protein population has unfolded. This confirms the protein is globally stable at and above physiological temperature (37°C), and is particularly important given the marginal thermal stability expected from the (GPP)₁₀ domain in the absence of hydroxylation.

How it is performed: Differential scanning fluorimetry (DSF, also known as ThermoFluor or protein thermal shift assay) was performed in a 96-well PCR plate format. Each well contained 18 µL of purified protein at 0.1–0.5 mg/mL in PBS and 2 µL of 100× SYPRO Orange dye (Thermo Fisher S6651; working concentration 5×). SYPRO Orange is environmentally sensitive and fluoresces strongly when bound to the hydrophobic core of unfolded proteins but is quenched in aqueous environments. Plates were sealed, briefly centrifuged, and loaded into a Bio-Rad CFX96 real-time PCR system. A temperature gradient from 25°C to 95°C was applied at a ramp rate of 0.5°C per 30 seconds, with fluorescence measured at each step using the FRET channel (excitation 490 nm, emission 575 nm). Raw fluorescence data were exported and processed in the DSFworld online tool or in GraphPad Prism. The melting temperature Tm was determined as the minimum of the first derivative (−dF/dT) of the melting curve. Experiments were performed in triplicate.

Technologies used: Differential scanning fluorimetry (DSF/ThermoFluor), real-time PCR thermocycler (Bio-Rad CFX96), SYPRO Orange dye, first-derivative curve analysis

Pass criterion: Tm ≥ 37°C, confirming the protein does not unfold at body temperature. Given that the (GPP)₁₀ domain has a predicted Tm of approximately 30–35°C without hydroxyproline and ≥ 40°C with hydroxyproline (Hyp), the DSF result directly reports whether HEK293T P4H activity was sufficient. A Tm below 37°C would constitute a failure of the collagen folding aim and would require either extended (GPP) repeat length or confirmed P4H co-expression.

Question 3: Technologies used — detailed descriptions

1. Sanger sequencing

Sanger (chain-terminator) sequencing is based on the selective incorporation of dideoxynucleotides (ddNTPs) during in vitro DNA synthesis. In the modern fluorescent variant, each of the four ddNTPs carries a different fluorophore. During PCR, the polymerase randomly incorporates a ddNTP instead of a dNTP, terminating chain elongation at that position. This produces a population of fragments of every possible length, each terminated by a fluorescently labelled base. These fragments are separated by capillary electrophoresis — passing through a polymer-filled capillary under an electric field — and detected by a laser as they elute. The output is a chromatogram where each peak position represents a nucleotide position and the colour of the peak identifies the base (A, T, G, or C). Read lengths of 700–900 bp are routinely achieved with high accuracy (>99.9% per base). In this project, Sanger sequencing confirms the identity of the codon-optimised insert at single-base resolution.

2. Agarose gel electrophoresis

Agarose gel electrophoresis separates nucleic acid molecules by size. Agarose — a polysaccharide derived from seaweed — forms a porous matrix when cast in buffer. When an electric field is applied, negatively charged DNA (due to its phosphate backbone) migrates toward the positive electrode. Smaller fragments migrate faster through the matrix than larger ones, creating separation by size over time. A fluorescent intercalating dye (GelRed or ethidium bromide) is incorporated into the gel or loading buffer and intercalates between base pairs; these dye-DNA complexes fluoresce brightly under UV light, allowing bands to be visualised. The size of each band is estimated by comparison to a DNA ladder — a mixture of DNA fragments of known sizes run in an adjacent lane. In this project, a 2% agarose gel resolves the ~282 bp insert released by HindIII/XhoI digest.

3. SDS-PAGE and western blotting

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins by molecular weight under denaturing conditions. SDS — a negatively charged detergent — binds to proteins in proportion to their mass, giving all proteins a uniform negative charge-to-mass ratio. Proteins are then driven through a polyacrylamide matrix by an electric field, with smaller proteins migrating faster. After electrophoresis, proteins are transferred from the gel to a PVDF membrane by applying an electric current perpendicular to the gel (wet or semi-dry transfer). The membrane is then probed with a primary antibody specific to an epitope on the target protein (in this case, the His₆ or FLAG tag), followed by a horseradish peroxidase (HRP)-conjugated secondary antibody that binds the primary. Addition of an ECL substrate causes HRP to catalyse a chemiluminescent reaction, and the emitted light is captured on film or a digital imager to reveal the position of the target protein as a band.

4. MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry determines the mass of intact molecules with high precision. The purified protein is co-crystallised with a light-absorbing organic matrix (typically CHCA or sinapinic acid) on a metal target plate. A short pulse from a UV laser ablates and ionises the matrix-protein co-crystals, transferring the energy to the protein without fragmenting it. The ionised proteins are accelerated through a vacuum tube by a high-voltage electric field. Because all ions receive the same kinetic energy, lighter ions travel faster and arrive at the detector first — their arrival time (time of flight) is directly proportional to their mass-to-charge ratio (m/z). For protein identification at the peptide level, tryptic digestion followed by LC-MS/MS provides sequence coverage and identifies sites of post-translational modification such as hydroxyproline (+15.9949 Da).

5. Circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy measures the differential absorption of left- and right-circularly polarised light by optically chiral molecules. Because the peptide bonds of proteins are chiral, and because different secondary structures (α-helix, β-sheet, random coil, polyproline II helix) position these bonds in distinct spatial arrangements, each secondary structure produces a characteristic CD spectrum in the far-UV region (190–260 nm). The polyproline type-II (PPII) helix characteristic of collagen triple helices produces a positive peak near 225 nm and a negative peak near 200 nm — a signature distinct from α-helices (which show two negative peaks at 208 and 222 nm) or disordered coils. The collagen triple helix is the specific target structure for the (GPP)₁₀ domain, as this conformation is required for piezoelectric dipole alignment. Thermal denaturation monitored by CD at 225 nm directly reports the melting temperature of this domain.

6. Fluorescence microscopy and Live/Dead assay

Fluorescence microscopy detects the emission of specific fluorescent molecules (fluorophores) after excitation with light of a defined wavelength. In the Live/Dead assay, two fluorophores with distinct spectral properties are used simultaneously. Calcein AM is a membrane-permeant dye that is cleaved by intracellular esterases in live cells to produce green-fluorescent calcein, which is retained in the cytoplasm — only metabolically active (live) cells produce a signal. Ethidium homodimer-1 (EthD-1) is a membrane-impermeant dye that can only enter cells with compromised plasma membranes (dead cells), where it intercalates into DNA and produces bright red fluorescence. Imaging cells stained with both dyes on a fluorescence microscope with appropriate filter sets simultaneously reveals the proportion of live (green) and dead (red) cells, enabling quantitative viability assessment.

7. Atomic force microscopy (AFM) nanoindentation and piezoresponse force microscopy (PFM)

AFM uses a sharp nanoscale tip (radius 2–50 nm) mounted on a flexible cantilever to interact with a sample surface. In nanoindentation mode, the tip is pressed into the sample surface with a controlled force, and the deflection of the cantilever (measured by a reflected laser beam) is recorded as a function of tip position. By fitting the resulting force-indentation curve to the Hertz contact mechanics model, the local Young’s modulus of the material is extracted. In piezoresponse force microscopy (PFM) mode, an alternating voltage is applied between the conductive tip and the grounded sample. If the material is piezoelectric, it locally deforms in phase with the applied AC voltage (converse piezoelectric effect), causing a detectable cantilever oscillation. The amplitude and phase of this oscillation, measured by lock-in detection, provide a nanoscale map of the piezoelectric response across the film surface — directly confirming the piezoelectric nature of the material at sub-micron resolution.

Summary table

Document prepared for HTGAA Final Project submission. Sequence: NQEQVSPL-(GGGGS)₃-GRGDS-IKVAV-(GPP)₁₀ | Vector: pcDNA3.1(+) | Host: HEK293T