CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity.

Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of  a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 µU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.

A Fully Integrated, Ready-to-Use Distance-Based Chemosensor for Visual Quantification of Multiple Heavy Metal Ions.

Point-of-care devices offering quantitative results with simple steps would allow great useability for untrained end-users. Here, we report a ready-to-use chemosensor integrating automatic sample metering, on-chip reaction, gravitational-magnetic separation, and a distance-based readout for visual quantification of multiple heavy metal ions. Deoxyribozymes (DNAzymes), probe-modified magnetic microparticles (MMPs), and polystyrene microparticles (PMPs) are preloaded into a microfluidic chip and freeze-dried. After the water sample is collected with automatic volume metering, the particles are resuspended, and the MMPs and PMPs hybridize with DNAzyme at its two termini, forming the "MMPs-DNAzyme-PMPs" structure. When target metal ions are present, the DNAzymes are cleaved, yielding an increased number of free PMPs. All on-chip reactions are controlled by stopping the liquid flow at capillary valves and bursting it with hand-controlled tilting. Using the chip with a gravitational-magnetic separator, the free PMPs are separated from "MMPs-DNAzyme-PMPs" and accumulate into the trapping channel with a nozzle, forming a visual bar with growing distances proportional to the concentration of target metal ions. The achieved limit of detection (LOD) values for Cu2+ (103.1 nM), Pb2+ (69.5 nM), and Ag+ (793.6 nM) are below the maximum contamination levels. High selectivity of 100-fold, 200-fold, and 20-fold against interference is obtained. Moreover, by integrating three identical channels in parallel, simultaneous detection of the above-mentioned heavy metal ions in fresh and tap water samples is also achieved with high accuracy. Together, this fully integrated and easily operated platform embodies excellent potential for rapid, on-site sensing by unskilled users.

Insertion of Pro-Hyp-Gly provides 2 kcal mol-1 stability but attenuates the specific assembly of ABC heterotrimeric collagen triple helices.

Collagen is a major structural component of the extracellular matrix and connective tissue. The key structural feature of collagen is the collagen triple helix, with a Xaa-Yaa-Gly (glycine) repeating pattern. The most frequently occurring triplet is Pro (proline)-Hyp (hydroxyproline)-Gly. The reversible thermal folding and unfolding of a series of heterotrimeric collagen triple helices with varying number of Pro-Hyp-Gly triplets were monitored by circular dichroism spectroscopy to determine the unfolding thermodynamic parameters Tm (midpoint transition temperature), ΔHTm (unfolding enthalpy), and ΔGunfold (unfolding free energy). The Tm and ΔGunfold of the heterotrimeric collagen triple helices increased with increasing number of Pro-Hyp-Gly triplets. The ΔGunfold increased by 2.0 ± 0.2 kcal mol-1 upon inserting one Pro-Hyp-Gly triplet into all three chains. The Tm difference between the most stable ABC combination and the second most stable BCC combination decreased with increasing number of Pro-Hyp-Gly triplets, even though the ΔGunfold difference remained the same. These results should be useful for tuning the stability of collagen triple helical peptides for hydrogel formation, recognition of denatured collagen triple helices as diagnostics and therapeutics, and targeted drug delivery.

Longer charged amino acids favor ß-strand formation in hairpin peptides.

Interactions between charged amino acids significantly influence the structure and function of proteins. The encoded charged amino acids Asp, Glu, Arg, and Lys have different number of hydrophobic methylenes linking the backbone to the charged functionality. It remains to be fully understood how does this difference in the number of methylenes affect protein structure stability. Protein secondary structures are the fundamental three-dimensional building blocks of protein structures. ß-Sheet structures are particularly interesting, because these structures have been associated with a number of protein misfolding diseases. Herein, we report the effect of charged amino acid side chain length at two ß-strand positions individually on the stability of a ß-hairpin. The charged amino acids include side chains with a carboxylate, an ammonium, or a guanidinium group. The experimental peptides, fully folded reference peptides, and fully unfolded reference peptides were synthesized by solid phase peptide synthesis and analyzed by 2D NMR methods including TOCSY, DQF-COSY, and ROESY. Sequence specific assignments were performed for all peptides. The chemical shift data were used to derive the fraction folded population and the folding free energy for the experimental peptides. Results showed that the fraction folded population increased with increasing charged amino acid side chain length. These results should be useful for developing functional peptides that adopt the ß-conformation.

Microfluidic particle accumulation for visual quantitation of copper ions.

A portable biosensor has been developed based on microfluidic particle accumulation for visual quantification of copper ions. A copper-dependent DNAzyme is used to connect magnetic microparticles (MMPs) and polystyrene microparticles (PMPs), forming "MMPs-DNAzyme-PMPs." When copper ions are present, the DNAzyme is cleaved, allowing free PMPs to be released from the MMPs-DNAzyme-PMP complex. Using a capillary-flow-based microfluidic device, the MMPs-DNAzyme-PMPs are first separated by a magnetic chamber, allowing the free PMPs to continue flowing until being trapped at a particle dam with a narrowing nozzle. Therefore, as a thermometer-like display, the copper level can be visually quantified by the accumulation length of the free PMPs in the trapping microchannel. The limit of detection (LOD) is 33 nM determined by the linear range of 25-100 nM, which is 900 times lower than the prevalent standard (~30 µM) in Hong Kong. The system shows excellent selectivity (> 1000-folds) against other heavy metal ions and abilities to adapt to multiple water environmental conditions. Tests on tap water samples and three local natural water sources in Hong Kong manifest that the device can effectively monitor the quality of freshwater with >70% recovery and 26.16% RSD.

Effects of Arginine Deimination and Citrulline Side-Chain Length on Peptide Secondary Structure Formation.

Post-translational modifications expand the chemical functionality of peptides and proteins beyond that originating from the encoded amino acids, but studies on the structural effects of these modifications have been limited. Arginine undergoes deimination to give citrulline (Cit), converting the positively charged guanidinium moiety into a neutral urea group. Herein, we report the effect of Arg deimination on secondary structure formation. To understand the reason for the number of methylene units in Cit, the effect of Cit side-chain length on secondary structure formation was also studied. Ala-based peptides and ß-hairpin peptides were used to study α-helix and ß-sheet formation, respectively. Peptides containing Cit analogues were prepared by an orthogonal protecting group strategy coupled with solid-phase carbamylation. The CD data for the Ala-based peptides were analyzed by using modified Lifson-Roig theory, showing that the helix propensity of Arg decreased upon deimination and that either shortening or lengthening Cit also decreased the helix propensity. The ß-hairpin peptides were analyzed by NMR methods, showing minimal change in strand formation energetics upon Arg deimination. Altering the Cit side-chain length did not affect strand formation energetics either. These results should be useful for the preparation of urea-bearing systems and the design of peptides incorporating urea-bearing residues with varying side-chain length.

Glycan-masking hemagglutinin antigens from stable CHO cell clones for H5N1 avian influenza vaccine development.

Refocusing of B-cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N-linked glycosylation motifs for glycan masking the vaccine antigen. We previously reported that glycan-masking recombinant H5 hemagglutinin (rH5HA) antigens on residues 83, 127, and 138 (g127 + g138 or g83 + g127 + 138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably expressing Chinese hamster ovary (CHO) cell clones for producing the glycan-masking g127 + g138 and g83 + g127 + g138 rH5HA antigens. All of these glycan-masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan-masking g127 + g138 but not g83 + g127 + g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem-binding antibodies, only the glycan-masking g127 + g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line-produced glycan-masking g127 + g138 rH5HA can be used for H5N1 subunit vaccine development.

Outline-etching image segmentation reveals enhanced cell chirality through intercellular alignment.

Cells cultured on micropatterns exhibit a chiral orientation, which may underlie the development of left-right asymmetry in tissue microarchitectures. To investigate this phenomenon, fluorescence staining of nuclei has been used to reveal such orientation. However, for images with high cell density, analysis is difficult because of the overlapping nuclei. Here, we report an image processing method that can acquire cell orientations within dense cell populations. After initial separation based on Boolean addition of binarized images using global and adaptive thresholds, the overlapping nucleus contours in the binarized images were segmented by iteratively etching the outlines of nuclei, which allowed the orientations of each cell to be extracted from densely packed cell clusters. In applying this technique to cultured C2C12 myoblasts in micropatterned stripes on different substrates, we found an enhanced chiral orientation on glass substrate. More important, this enhanced chirality was consistently observed with increased intercellular alignment and independent of cell-cell distance or cell density, suggesting that intercellular alignment plays a role in determining the chiral orientation. By segmenting single cells with intact orientation, this technique offers an automated method for quantitative analysis with improved accuracy, providing an essential tool for studying left-right asymmetry and other morphogenic dynamics in tissue formation.

Protein-Substrate Adhesion in Microcontact Printing Regulates Cell Behavior.

Microcontact printing (µCP) is widely used to create patterns of biomolecules essential for studies of cell mechanics, migration, and tissue engineering. However, different types of µCPs may create micropatterns with varied protein-substrate adhesion, which may change cell behaviors and pose uncertainty in result interpretation. Here, we characterize two µCP methods for coating extracellular matrix (ECM) proteins (stamp-off and covalent bond) and demonstrate for the first time the important role of protein-substrate adhesion in determining cell behavior. We found that, as compared to cells with weaker traction force (e.g., endothelial cells), cells with strong traction force (e.g., vascular smooth muscle cells) may delaminate the ECM patterns, which reduced cell viability as a result. Importantly, such ECM delamination was observed on patterns by stamp-off but not on the patterns by covalent bonds. Further comparisons of the displacement of the ECM patterns between the normal VSMCs and the force-reduced VSMCs suggested that the cell traction force plays an essential role in this ECM delamination. Together, our results indicated that µCPs with insufficient adhesion may lead to ECM delamination and cause cell death, providing new insight for micropatterning in cell-biomaterial interaction on biointerfaces.

Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design.

UNLABELLED: Influenza virus hemagglutinin (HA) protein consists of two components, i.e., a globular head region and a stem region that are folded within six disulfide bonds, plus several N-linked glycans that produce a homotrimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by an HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1 stem and HA2 stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2 stem region (H5 N484A and H1 N503A) was found to elicit more potent neutralizing antibody titers against homologous, heterologous, and heterosubtypic viruses. Unmasking the HA2 stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2 stem mutant protein immunization increased the numbers of antibody-secreting cells, germinal center B cells, and memory B cells targeting the stem helix A epitopes in splenocytes. Unmasking the HA2 stem N-glycans of H5HA mutant proteins showed a significantly improvement in the protection against homologous virus challenges but did so to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines. IMPORTANCE: N-linked glycosylation sites in the stem regions of influenza virus hemagglutinin (HA) proteins are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Our studies indicate that unmasking the HA2 stem N-glycans of recombinant HA proteins from H5N1 and pH1N1 viruses induced more potent neutralizing antibody titers against homologous and heterosubtypic viruses. However, only immunization with the H5N1 HA2 stem mutant protein can refocus B antibody responses to the helix A epitope for inducing more CR6261-like/FI6v3-like and fusion inhibition antibodies in antisera, resulting in a significant improvement for the protection against lethal H5N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

Visual detection of nucleic acids based on Mie scattering and the magnetophoretic effect.

Visual detection of nucleic acid biomarkers is a simple and convenient approach to point-of-care applications. However, issues of sensitivity and the handling of complex bio-fluids have posed challenges. Here we report on a visual method detecting nucleic acids using Mie scattering of polystyrene microparticles and the magnetophoretic effect. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) were surface-functionalised with oligonucleotide probes, which can hybridise with target oligonucleotides in juxtaposition and lead to the formation of MMPs-targets-PMPs sandwich structures. Using an externally applied magnetic field, the magnetophoretic effect attracts the sandwich structure to the sidewall, which reduces the suspended PMPs and leads to a change in the light transmission via the Mie scattering. Based on the high extinction coefficient of the Mie scattering (∼3 orders of magnitude greater than that of the commonly used gold nanoparticles), our results showed the limit of detection to be 4 pM using a UV-Vis spectrometer or 10 pM by direct visual inspection. Meanwhile, we also demonstrated that this method is compatible with multiplex assays and detection in complex bio-fluids, such as whole blood or a pool of nucleic acids, without purification in advance. With a simplified operation procedure, low instrumentation requirement, high sensitivity and compatibility with complex bio-fluids, this method provides an ideal solution for visual detection of nucleic acids in resource-limited settings.

Branching patterns emerge in a mathematical model of the dynamics of lung development.

Recent experimental work has described an elegant pattern of branching in the development of the lung. Multiple forms of branching have been identified, including side branching and tip bifurcation. A particularly interesting feature is the phenomenon of 'orthogonal rotation of the branching plane'. The lung must fill 3D space with the essentially 2D phenomenon of branching. It accomplishes this by rotating the branching plane by 90° with each generation. The mechanisms underlying this rotation are not understood. In general, the programmes that underlie branching have been hypothetically attributed to genetic 'subroutines' under the control of a 'global master routine' to invoke particular subroutines at the proper time and location, but the mechanisms of these routines are not known. Here, we demonstrate that fundamental mechanisms, the reaction and diffusion of biochemical morphogens, can create these patterns. We used a partial differential equation model that postulates three morphogens, which we identify with specific molecules in lung development. We found that cascades of branching events, including side branching, tip splitting and orthogonal rotation of the branching plane, all emerge immediately from the model, without further assumptions. In addition, we found that one branching mode can be easily switched to another, by increasing or decreasing the values of key parameters. This shows how a 'global master routine' could work by the alteration of a single parameter. Being able to simulate cascades of branching events is necessary to understand the critical features of branching, such as orthogonal rotation of the branching plane between successive generations, and branching mode switch during lung development. Thus, our model provides a paradigm for how genes could possibly act to produce these spatial structures. Our low-dimensional model gives a qualitative understanding of how generic physiological mechanisms can produce branching phenomena, and how the system can switch from one branching pattern to another using low-dimensional 'control knobs'. The model provides a number of testable predictions, some of which have already been observed (though not explained) in experimental work.

Left-right symmetry breaking in tissue morphogenesis via cytoskeletal mechanics.

RATIONALE: Left-right (LR) asymmetry is ubiquitous in animal development. Cytoskeletal chirality was recently reported to specify LR asymmetry in embryogenesis, suggesting that LR asymmetry in tissue morphogenesis is coordinated by single- or multi-cell organizers. Thus, to organize LR asymmetry at multiscale levels of morphogenesis, cells with chirality must also be present in adequate numbers. However, observation of LR asymmetry is rarely reported in cultured cells. OBJECTIVES: Using cultured vascular mesenchymal cells, we tested whether LR asymmetry occurs at the single cell level and in self-organized multicellular structures. METHODS AND RESULTS: Using micropatterning, immunofluorescence revealed that adult vascular cells polarized rightward and accumulated stress fibers at an unbiased mechanical interface between adhesive and nonadhesive substrates. Green fluorescent protein transfection revealed that the cells each turned rightward at the interface, aligning into a coherent orientation at 20° relative to the interface axis at confluence. During the subsequent aggregation stage, time-lapse videomicroscopy showed that cells migrated along the same 20° angle into neighboring aggregates, resulting in a macroscale structure with LR asymmetry as parallel, diagonal stripes evenly spaced throughout the culture. Removal of substrate interface by shadow mask-plating, or inhibition of Rho kinase or nonmuscle myosin attenuated stress fiber accumulation and abrogated LR asymmetry of both single-cell polarity and multicellular coherence, suggesting that the interface triggers asymmetry via cytoskeletal mechanics. Examination of other cell types suggests that LR asymmetry is cell-type specific. CONCLUSIONS: Our results show that adult stem cells retain inherent LR asymmetry that elicits de novo macroscale tissue morphogenesis, indicating that mechanical induction is required for cellular LR specification.

Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

Theranostic DNA nanostructure based on phenotype-specific activation of antisense oligonucleotides.

Antisense oligonucleotide (ASO) is a powerful agent for gene therapy, designed to form complementary pairs with specific mRNA to inhibit gene expression. However, low specificity limits its potential. To overcome this challenge, we developed a Y-shape DNA nanostructure that enhances the specificity in ASO-based treatment by introducing a detection trigger. The design incorporates the phenotype-specific miR21 activation and the sequential release of Bcl2 ASO. As a result, our Y-shape DNA nanostructure downregulates >50 % Bcl2 mRNA expression and induces >60 % cell death in breast cancer cells. Meanwhile, this approach shows no obvious damage to the non-cancerous cells, indicating the therapeutic potential as a theranostics agent in precision medicine with the combination of biomarker sensing and treatment. Overall, our Y-shape DNA nanostructure serves as a promising strategy providing potential in customized conformation design with specific target sequences in gene therapy.

On-Site Melanoma Diagnosis Utilizing a Swellable Microneedle-Assisted Skin Interstitial Fluid Sampling and a Microfluidic Particle Dam for Visual Quantification of S100A1.

Malignant melanoma (MM) is the most aggressive form of skin cancer. The delay in treatment will induce metastasis, resulting in a poor prognosis and even death. Here, a two-step strategy for on-site diagnosis of MM is developed based on the extraction and direct visual quantification of S100A1, a biomarker for melanoma. First, a swellable microneedle is utilized to extract S100A1 in skin interstitial fluid (ISF) with minimal invasion. After elution, antibody-conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are introduced. A high expression level of S100A1 gives rise to a robust binding between MMPs and PMPs and reduces the number of free PMPs. By loading the reacted solution into the device with a microfluidic particle dam, the quantity of free PMPs after magnetic separation is displayed with their accumulation length inversely proportional to S100A1 levels. A limit of detection of 18.7 ng mL-1 for S100A1 is achieved. The animal experiment indicates that ISF-based S100A1 quantification using the proposed strategy exhibits a significantly higher sensitivity compared with conventional serum-based detection. In addition, the result is highly comparable with the gold standard enzyme-linked immunosorbent assay based on Lin's concordance correlation coefficient, suggesting the high practicality for routine monitoring of melanoma.

Spectrally engineered textile for radiative cooling against urban heat islands.

Radiative cooling textiles hold promise for achieving personal thermal comfort under increasing global temperature. However, urban areas have heat island effects that largely diminish the effectiveness of cooling textiles as wearable fabrics because they absorb emitted radiation from the ground and nearby buildings. We developed a mid-infrared spectrally selective hierarchical fabric (SSHF) with emissivity greatly dominant in the atmospheric transmission window through molecular design, minimizing the net heat gain from the surroundings. The SSHF features a high solar spectrum reflectivity of 0.97 owing to strong Mie scattering from the nano-micro hybrid fibrous structure. The SSHF is 2.3°C cooler than a solar-reflecting broadband emitter when placed vertically in simulated outdoor urban scenarios during the day and also has excellent wearable properties.

Microfluidic particle counter visualizing mucosal antibodies against SARS-CoV-2 in the upper respiratory tract for rapid evaluation of immune protection.

Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL-1; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL-1; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.

Single-Cell Micropatterning by Non-fouling Hydrogels.

Micropatterned substrate is a unique method for studying cell biology at the single-cell level. Using photolithography to create binary patterns of cell-adherent peptide surrounding by non-fouling cell-repellent poly(ethylene glycol) (PEG) hydrogel, this patterning method allows for controlling cell attachment with desired sizes and shapes up to 19 days. Here we provide the detailed procedure of fabrication for such patterns. This method will allow monitoring of prolonged reaction of single cells such as cell differentiation upon induction or time-resolved apoptosis stimulated by drug molecules for cancer treatment.