1. NanoCone Technology for Bioanalysis and Medical Diagnostics
NanoCone is an alias of dendrons, conically shaped organic macromolecules, and belongs to the dendrimer family of which shape is globular mostly. We designed the dendron so that there is a single active group at the apex for the conjugation with the capture probes such as DNA and antibodies, and there are multiple groups at the periphery that bind to solid substrates such as glass slides and silicon wafers. Also judicious selection of the backbone minimizes the nonspecific binding of straying biomolecules. It is possible to assemble a monolayer of NanoCone on the substrates through dipping processes, and the processes are mass-production compatible (Fig. 1). Because periphery of the dendron occupies a certain area of the surface, it is possible to generate spacing between the active groups at the apex. With the second and third generation dendrons, lateral spacing of 3 nm and 6 nm can be realized on surface, respectively. After conjugating tiny gold nanoparticles at the apex of the self-assembled second generation dendron, high resolution SEM images clearly showed that the most probable lateral spacing was 3.2 nm (Fig. 2) The surface of 3 nm is suitable to immobilize DNAs, while the surface 6 nm is good for proteins such as antibodies. Because the diameter of DNA duplex is 2.5 nm, and width of antibody is about 5-6 nm, the capture DNA and antibody can secure ample space despite of the surface confinement. Certainly, the former surface turned to be good for DNA microarrays, and the latter surface protein microarrays. In particular, a few investigators in the campus participated in further characterization of the surface, and found unusually high thermal stability of DNA probes on NanoCone surface. The observation brought a new idea to combine microarrays and PCR. In other words, a glass rod on which end a few DNA spots were fabricated was placed in a PCR tube, and after PCR process the captured target DNAs could be imaged right after PCR (Analytical Biochemistry, (2010) 139). Whereas the initial effort was given to demonstrate efficacy of DNA microarrays on NanoCone surface, the continuous studies indicate that there are numerous applications including antibody microarrays. Use of the surface for atomic force microscope is a stimulating example.
Figure 1 Figure 2
Commercialization: The approach has been applied for various substrates including glass slides, glass rods, silicon wafers, membranes, and others. Up to now, tens of thousands glass slides and hundreds of thousands glass rods have been produced and sold to companies and researchers in schools and research institutes world-wide.
Patent: 1) “Size-Controlled Macromolecule” US 9201067 B2, US 9389227 B2, US 9671396 B2외 CN, JP, KR, CA, RU, AU, IN, DE, CH, FR, GB registered
Publication: 1) J. W. Park et al., "Self-Assembly of a Dendron through Multiple Ionic Interaction to Give Mesospacing between Reactive Amine Groups on the Surface" Langmuir, (2003) 2357. 2) J. W. Park et al., “Enhanced Protein-Ligand Interactions by Guaranteed Mesospacing between Immobilized Biotins”, Chem. Commun., (2004) 1316. 3) J. W. Park et al., “Nanoscale-Controlled Spacing Provides DNA Microarrays with the SNP Discrimination Efficiency in Solution Phase” Langmuir, (2005) 4257. 4) J. W. Park et al., “Spatially Nanoscale-Controlled Functional Surfaces Toward Efficient Bioactive Platforms” J. Materials Chemistry B, (2015) 5135.
2. Metal Enhanced Fluorescence Nanoparticle
Great attention has been paid to increase the fluorescence intensity from an analyte, because such increase makes bioanalysis more sensitive and allows use of less sensitive (or less expensive) readers. Typically about five fluorescence tags are conjugated at an antibody to enhance the signal intensity. Incorporating much higher number of fluorescence tags in a bead looks an alternative for the larger signal, but quenching of the excited state by tags at the vicinity makes the approach marginable better than the former. However, Dr. J.-M. Nam (the inventor, a professor of Seoul National University) placed the tags at a specific distance from the metal surface, and utilized the strong plasmon field at both ends of the rod-shaped nanoparticle. He demonstrated that the intensity increases by a factor of 15,700 times at 647 nm (Fig. 3). Application of such nanoparticles seems unlimited. When the particles are used for the microarrays, the limit of detection (LOD) is enhanced by a factor of more than 10,000, and the approach is very attractive when target biomolecules are hard to amplify. Also, eliminating use of enzymes for ELISA can be envisioned, and even higher sensitivity is expected.
Commercialization: On the way for the large scale synthesis
Patent: 1) “Core-Shell nanocomposite for metal-enhanced fluorescence” KR 10-1597894 B1 registered, EP 3272831 A1 granted, US 2018-0044584 A1 published.
Publication: Professor Jwa-Min Nam (the inventor, a professor of Seoul National University) is a world-wide famous expert for the nanoparticles, and published many articles in prestigious journals such as Nature Materials, J. Am. Chem. Soc., and others.
3. Lateral Flow Assay Kits – Toward LOQ of 0.1 ng/ml Troponin I
Lateral flow assay kits are widely used for screening various diseases, and world-wide annual sale of 7 B USD reflects the popularity and acceptance in the medical society. The kit consists of sample pad, conjugation pad, membrane, and absorbent pad (Fig. 4), and nitrocellulose membrane is the most commonly used membrane, and various types are available commercially. On the membrane capture antibodies are placed on the test line, and generic antibodies are sprayed on the control line. After allowing a sample (whole blood or blood plasma) to move along the membrane through micro-capillary action, signal intensities of the test line and the control line are recorded at a reader, and the ratio gives concentration of the target protein of interest quantitatively or qualitatively. Cost-effectiveness and convenience are the major advantages of the kit, but because the capture antibody is immobilized on the membrane through the non-specific binding, and because surface of the membrane is irregular commonly, the limited reproducibility is a weak part of the kit. For example, typically LOQ (the lowest concentration reaching at 10% CV) is 0.5 ng/ml Troponin I at the most favorable condition.
We are pleased to note that LOQ reaches 0.1 ng/ml Troponin I, when NanoCone technology is applied to nitrocellulose membrane. Whereas for easier shipping and storage of the kits, the kit of all-dry-state is desirable, such LOQ moves to 0.3 ng/ml. Because the cardiac society concurs that it is necessary to screen down to 0.1 ng/ml Troponin I to find whether he or she is not at risk of cardiac infarction, manufacturing LFA kits sensitive enough to make LOQ of 0.1 ng/ml Troponin I is a highly motivated technical goal (Fig. 5). In order to meet the medical unmet need, we employed both NanoCone technology and metal enhanced fluorescence nanoparticles, and observed LOQ of 0.1 ng/ml. Now, we are optimizing the condition for the large scale synthesis of the nanoparticles in collaboration with Professor Nam.
Figure 4 Figure 5
Commercialization: A pilot production facility was completed in 2017, and the current maximum production capacity is 50 M kits per year. ISO 13485 was obtained in 2018, and CE mark for the kits of five cardiac biomarkers will be given in 2019.
Patent: 1) “Method for improving quantitative properties of nitrocellulose membrane using organic nanostructured molecule” PCT applied (2018).
4. DNA and Protein Microarrays – Enhanced LOD by a Factor of 1,000
Initially the main effort for the application went to DNA microarrays. As a result, it was demonstrated that NanoCone-coated surface promises fast kinetics of the binding event and enhanced discrimination of Single Nucleotide Polymorphism, certainly because each probe immobilized on surface can enjoy ample space for the fast binding. On top of this, the melting temperature difference between the matched and mismatched pairs is noticeable, because the surface bound effect is minimal. For example, after washing, the intensity of a micro-arrayed spot is about 1 % when the corresponding target DNA has the single point mutation internally. Fabricating numerous spots in a microarray is common, and because the microarray has to go through hybridization and washing at selected temperatures, it is important to design the capture probes so that melting temperature of the expected duplexes has to be within a narrow temperature window. Nevertheless, the real melting temperature frequently deviates from the calculated one due to the surface confinement effect. NanoCone surface minimizes such deleterious alteration so that selected washing and hybridization temperatures work well for all spots in a microarray.
Recently, about 20 antibodies were selected to fabricate a phosphokinase antibody microarray, and it was demonstrated that such microarray works nicely for analyzing regulated proteins in brain tissue samples from Alzheimer’s disease model mice (Plos One, 2014).
More recently, combination of such substrates and metal enhanced fluorescence nanoparticles was employed for better performance microarrays for quantifying microRNAs. It was observed that the LOD is lower than 1 fM, and thus enhancement of more than 1,000 times was recorded.
Commercialization: Microarrays requiring high sensitivity are target products in the near future, and partnering with companies specializing microarrays should be a good strategy for prompt market penetration and early success of the business. It should be possible to make ELISA kits in which not only use of the enzymes is not required, but also highly enhanced sensitivity is guaranteed.
Patent: 1) “Size-Controlled Macromolecule” US 9201067 B2, US 9389227 B2, US 9671396 B2외 CN, JP, KR, CA, RU, AU, IN, DE, CH, FR, GB registered. 2) “Polynucleotide synthesis on a modified surface” US 8647823.
Publication: 1) J. W. Park et al., “Nanoscale-Controlled Spacing Provides DNA Microarrays with the SNP Discrimination Efficiency in Solution Phase” Langmuir, (2005) 4257. 2) J. W. Park et al., “DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene” Nucleic Acids Research, (2005) e90. 3) J. W. Park et al., “DNA Microarrays on Nanoscale-Controlled Surface”, Nucleic Acids Research, (2005) e106. 4) J. W. Park et al., “Phosphokinase Antibody Arrays on a Dendron-Coated Surface”, Plos One, (2014) e96456. 5) J. W. Park et al., “Surface Modification for DNA and Protein Microarrays", Omics, (2006) 327. 6) J. W. Park et al., “Biofunctional Dendrons Grafted on a Surface” In “Handbook of Biofunctional Surfaces” Pan Stanford Publishing Pte. Ltd., USA (2013). + others
5. Atomic Force Microscopy – A New Approach Reaching the Ultimate Sensitivity for Biomarker Quantification
Atomic Force Microscope is a younger sister of scanning tunneling microscope, and the former microscope senses the force between atoms (atoms on a substrate and atoms at the probe) with high lateral resolution (0.1 nm). The microscope was evolved to measure the interaction force between biomolecules in solution phase, and was utilized to investigate the folding of proteins and the behavior of target molecules in a cell. The team of Professor J. W. Park observed that use of NanoCone coating on the probes and substrates guarantees 1:1 interaction between the immobilized DNAs. Therefore, they were able to enhance the reproducibility and precision of the measurement (Fig. 6). They demonstrated that the approach is applicable to other biomolecules such as RNAs and proteins, and it is possible to count the copy number of the captured biomarkers on surface by scanning. By employing a tiny spot such as 1-2 nm in diameter, it is possible to scan the whole spot area and count the copy number of a translocated DNA biomarker down to single copy without amplification or labeling. As far as protein biomarkers are concerned, they showed that LOD is good enough to quantify the biomarker in a single cell. It is expected to enhance the LOD as far as the nonspecific binding of straying proteins is reduced further to lower the background signal. As a result, AFM is newly reborn as a bioanalytical tool, and the sensitivity reaches single copy of DNA biomarkers and a few hundreds of protein biomarkers (Fig. 7). Now, the team is keen on counting copy number of mutated or methylated genes in a drop of blood samples (liquid biopsy) without amplification. We certainly believe the approach gives great impact on bioanalytical and diagnostic fields due to the unprecedented sensitivity and wide applicability.
Figure 6 Figure 7
Commercialization: The initial market goes to the research area, automation and simplification of the tool as well as availability of relevant computer programs to treat raw data should help wide acceptance of the approach. Also, fast turn-over time will cut down cost of the analysis for each sample. Due to new AFMs of faster speed have been commercialized recently, it will be possible to complete the analysis in a few minutes rather than 1 hour. After being established in the research market, AFM will be able to move to the medical diagnostics market because of the unprecedented sensitivity.
Patent: 1) “Biomolecule interaction using atomic force microscope” US 8673621 B2, CN 101322030, JP 4753392, KR 10-1069898, KR 10-69899, US 9175335 B2. 2) “Atomic force microscope as an analyzing tool for biochip” US 2009-0048120 A1. 3) “마이크로 RNA 초민감성 정량분석 기술 (ultrasensitive quantification of microRNAs)” KR 10-1742211 B1, US 2017/0233799. 4) “단일세포 내 마이크로 RNA의 고해상도 시각화 기술 (high resolution visualization of microRNAs in a single cell)” KR 10-17377450. 5) “Method and apparatus for ultrasensitive quantitative analysis of DNA biomarker” US 2018-0135111 A1.
Publication: 1) J. W. Park et al., “Dendron Arrays for the Force-Based Detection of DNA Hybridization Events”, J. Am. Chem. Soc., (2007) 9349. 2) J. W. Park et al., ““Seeing and Counting” Individual Antigens Captured on a Microarrayed Spot with Force-Based Atomic Force Microscopy”, Anal. Chem., (2010) 5189. 3) J. W. Park et al., “Direct Quantitative Analysis of HCV RNA by Atomic Force Microscopy without Labeling or Amplification”, Nucleic Acids Research, (2012) 11728. 4) J. W. Park et al., “Spatially Nanoscale-Controlled Functional Surfaces Toward Efficient Bioactive Platforms” J. Materials Chemistry B, (2015) 5135. 5) J. W. Park et al., “Quantification of Fewer Than Ten Copies of a DNA Biomarker without Amplification or Labeling”, J. Am. Chem. Soc., (2016) 7075. 6) J. W. Park et al., "Direct quantification of trace amounts of a chronic myeloid leukemia (CML) biomarker using locked nucleic acid capture probes", Anal. Chem., (2018) 12824. 7) J. W. Park et al., “Ultra-sensitive and Label-Free Probing of Binding Affinity Using Recognition Imaging”, Nano Letters, (2019) 612. + others