Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in singlemolecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multiconditional and multiparametric assays, hindering in-depth understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a digital bioassay platform with easy solution exchanges, called multidimensional (MD) digital bioassay. We immobilized single analytes in arrayed femtoliter (10 −15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. With multiconditional assays based on our system, we could quantitatively determine inhibitor sensitivities of single influenza A virus particles and single alkaline phosphatase (ALP) molecules, which has never been achieved with conventional digital bioassays. Further, we demonstrated that ALPs from two origins can be precisely distinguished by a single-molecule multiparametric assay with our system, which was also difficult with conventional digital bioassays. Thus, MD digital bioassay is a versatile platform to gain in-depth insight into biological entities in unprecedented resolution.

Abstract— A blue‐light‐emitting Eu2+‐doped CaMgSi2O6 phosphor having a long lifetime for a plasma‐display panel (PDP) was developed. The CaMgSi2O6:Eu2+(CMS:Eu) phosphors show no luminance degradation during the baking process, and an equivalent photoluminescence peak intensity compared to that of the conventional blue‐phosphor BaMgAl10O17:Eu2+ (BAM) after baking. CMS: Eu shows a poor luminescent characteristic for the Xe excimer band excitation due to the lack of absorption. To introduce the absorption center for the Xe excimer band, we performed Gd‐codoping of CMS: Eu as a sensitizer and found a new excitation band around 172 nm, which originated from Gd3+. The test PDPs panels using synthesized CMS: Eu phosphor and CMS: Eu, Gd phosphor were examined to investigate the luminescent and aging characteristics of a Xe‐discharge excitation source. The CMS: Eu panel shows an emission peak intensity comparable to that of the BAM panel (i.e., a comparable stimuli L/CIEy, 93% of BAM), while the CMS: Eu, Gd panel shows poorer blue emission intensity compared to the BAM panel (up to 53% of total stimuli of BAM). The CMS: Eu panel and the CMS: Eu, Gd panel show less luminance degradation than the BAM panel under the aging test, and the panel retains 90% of its luminance after 300 hours of driving. It was found that CMS: Eu appears to be a candidate for a new blue PDP phosphor because of its longevity in a Xe‐discharge plasma environment.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multi-conditional or multi-parametric assays, hindering understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a new digital bioassay platform with easy solution exchanges, called multi-dimensional (MD) digital bioassay, and tested its quantitativity and utility. We immobilized single analytes in arrayed femtoliter (10-15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. To show the utility of our system, we investigated neuraminidase inhibitor (NAI) sensitivity on single influenza A virus (IAV) particles in a multi-conditional assay. We proved that IAV particles show a heterogeneous response to the NAI. Further, to demonstrate multi-parametric assays, we examined the sensitivity of individual IAV particles or model enzyme molecules to two different inhibitors. Our results support that MD digital bioassay is a versatile platform to unveil heterogeneities of biological entities in unprecedented resolution.