Figure 2. HRSEM of microwells on TiN biochip

For PCR, it uses fluorescent dye as the optical transducer and the dye molecules are liberated from quenchers on target-receptor binding to produce signal. The advantage of PCR is that it is well-known with proven record of performance. However, the limitations of PCR are also obvious.

• The number of dyes used in a single measurement is rather limited. As the number of dyes increases, color crosstalk also increases making it difficult to draw conclusion on  multiplex measurement.
• PCR needs polymerase enzymes to build the complementary DNA/RNA chains from the forward and reverse primer. Then the newly built DNA/RNA helices are thermally degenerated for further replication. Unfortunately, the build-degenerate-replicate process is not error free.
• The PCR test result is always qualitative as it measures the replicated copies of DNA/RNA target with polymerase and thermal cycling. It never tells the absolute number of copies of the DNA/RNA target initially present in the sample.

Although existing SPR systems are free from PCR limitations, they have issues on their own. Some of these are so significant that it deters SPR becoming the major player for in-vitro diagnostics (IVD).

• Gold is the sole material in commercial SPR biosensors. It is very expensive raw material as gold price doubled in a decade.
• Freshly prepared thiol based chemicals are necessary to activate the gold surface to enable biosensing. This is a critical step and the thiol linker is unstable, so it complicates the operation protocol.  
• The gold film deposited for SPR biosensing requires stringent thickness to 50 nm with precision of 1 nm. Either too thick or too thin gold film will reduce the SPR sensitivity.
• There is a fundamental dilemma with SPR measurement principle. For the best sensitivity, it is desirable to have optimal resonance by optical excitation. However, if incident light is totally transferred to collective electron cloud oscillation, the measurment is prone to poor signal-to-noise ratio.

In order to circumvent the issue, commercial SPR systems measure the light intensity away from optimal resonance to collection meaningful data but the sensitivity suffers.

Figure 3. High magnificant HRSEM of our biochip

To address limitations of exisiting SPR systems, we invented the next generation plasmonic biosensor based on TiN nanocubes. A high magnification HRSEM image of our biochip is shown in Figure 3. Our game-changing TiN plasmonic technology delivers:

1. Massive parallel analysis upto 144 receptors against a single target in one run,
2. Replacement of gold thin film by synthetic titanium nitride nanocubes about 45nm in size,
3. Direct hybridization of DNA/RNA complementary pairs with thermoplasmonic effect,
4. Simplified functionalization protocol by biotinylation and chemisorption,
5. Measurement of plasmonic enhanced elastic scattering instead of attenuated total internal reflection thus better signal-to-noise ratio,
6. Flexible manufacturability by 3D printing.

Figure 4. Distribution of Ti and N on biochip surface

With these achievements, we have applied and granted invention patents for our TiN technology in China and US. To further illustrate the distribution of TiN nanocubes on the biochip surface, we performed high-resolution scanning electron mictoscopy X-ray energy dispersive analysis (HRSEM-EDX) in the smallest scale of 500 nanometer. The distribution in Figure 4 shows that Ti and N are present with even distribution.

To investigate the surface chemistry for functionalization of TiN biochip, we use our in-house super-computing cluster to calculate the adsorption energy of various potential candidate molecules on TiN(200) surface by density functional theory (DFT) [3] and standard pseudo-potential library [4]. Open-sourced DFT code Quantum-Espresso v6.7 [5] was used for all calculation. We are particular interested to see binding of oligonucleotides on TiN surface i.e., Adenine (A), Guanine (G), Cytosine (C), Uracil (U). The simulation shows that the PO4 functional group at the 5' of these nucleotides is the preferred anchoring position on TiN(200) with the most negative adsorption energy. Whereas the most unstable orientation is via the base at position 1'. The adsorption energy difference is so pronounced that it exceeds 2eV. Therefore we believe that the adsorption via the PO4 group at 5' is covalent, whereas the adsorption via the base at 1' is van der Waals forces. The discovery is actually beneficial to DNA/RNA sequences anchored on the TiN surface for biosensing purpose. This is due to the hybridization of DNA/RNA always take place via hydrogen bonds at the base position at 1'. So, anchoring via the 5' position, it does not compete with hybridization of complimentary base pairs. Details of our DFT computation steps can be found on the simulation page.

With the above ground breaking discoveries, we believe the TiN biosensors shall bring about label-free revolution to the IVD market worldwide.

References

[1] Surface Plasmon Resonance (SPR), https://en.wikipedia.org/wiki/Surface_plasmon_resonance

[2] Polymerase Chain Reaction (PCR), https://en.wikipedia.org/wiki/Polymerase_chain_reaction

[3] Density functional theory (DFT), https://en.wikipedia.org/wiki/Density_functional_theory

[4] Standard solid-state pseudopotentials, https://www.materialscloud.org/discover/sssp/table/efficiency

[5] Quantum ESPRESSO, https://www.quantum-espresso.org/

China invention patents filed and granted: