Small-molecule Sensors Research Articles

Atomistic Simulations Reveal Crucial Role of Metal Ions for Ligand Binding in Guanidine-I Riboswitch.

Riboswitches are structured ribonucleic acid (RNA)segments that act as specific sensors for small molecules in bacterial metabolism. Due to the flexible nature of these highly charged macromolecules, molecular dynamics simulations are instrumental to investigating the mechanistic details of their regulatory function. In the present study, the guanidine-I riboswitch serves as an example of how atomistic simulations can shed light on the effect of ions on the structure and dynamics of RNA and on ligand binding. Relying on two orthologous crystal structures from different bacterial species, it is demonstrated how the ion setup crucially determines whether the simulation yields meaningful insights into the conformational stability of the RNA, functionally relevant residues and RNA-ligand interactions. The ion setup in this context includes diffuse ions in solution and bound ions associated directly with the RNA, in particular a triad of 2 Mg2+ ions and a K+ ion in close proximity to the guanidinium binding site. A detailed investigation of the binding pocket reveals that the K+ from the ion triad plays a decisive role in stabilizing the ligand binding by stabilizing important localized interactions, which in turn contribute to the overall shape of the folded state of the RNA.

Historic obstacles and emerging opportunities in the field of developmental metabolism - lessons from Heidelberg.

The field of developmental metabolism is experiencing a technological revolution that is opening entirely new fields of inquiry. Advances in metabolomics, small-molecule sensors, single-cell RNA sequencing and computational modeling present new opportunities for exploring cell-specific and tissue-specific metabolic networks, interorgan metabolic communication, and gene-by-metabolite interactions in time and space. Together, these advances not only present a means by which developmental biologists can tackle questions that have challenged the field for centuries, but also present young scientists with opportunities to define new areas of inquiry. These emerging frontiers of developmental metabolism were at the center of a highly interactive 2023 EMBO workshop 'Developmental metabolism: flows of energy, matter, and information'. Here, we summarize key discussions from this forum, emphasizing modern developmental biology's challenges and opportunities.

Fast fluorescence sensing of aflatoxin B1 employing a europium metal–organic framework avoiding self-fluorescence interference of target

Aflatoxin B1 (AFB1) easily contaminates grain, oil, and their products, and is strongly carcinogenic. Therefore, rapid, efficient and sensitive detection of aflatoxin plays a key role in the food industry. Here, we prepared a lanthanide metal-organic framework termed Eu-TCPP, in which the ligand can effectively sensitize Eu3+ with excellent photoluminescence properties. It is worth mentioning that the fluorescence intensity of Eu-TCPP is at 617 nm, which can avoid the interferences of AFB1 self-fluorescence emitting in the range of 400 nm–500 nm. Eu-TCPP can efficiently detect AFB1 without using antibody or biomolecular modifications, exhibiting a rapid response of 60 seconds and a limit of detection (LOD) at 11.63 ppb. The Eu-TCPP sensor also has satisfactory performance for determining AFB1 sensitively in real oil samples. In addition, the mechanism of the excellent detection performance of Eu-TCPP has been investigated using density-functional theory (DFT) calculations combined with experimental analysis. The results show that the LUMO energy level of the ligand is higher than that of the aflatoxin, which causes the electrons transfer toward the targets, and results in the reduction of the fluorescence intensity. Therefore, our proposed Eu-MOF materials based on the "antenna effect" have great potential for the development of simple, fast, and sensitive small-molecule sensors, and offer a promising prospect in POCT applications.

BODIPYs α-appended with distyryl-linked aryl bisboronic acids: single-step cell staining and turn-on fluorescence binding with D-glucose.

Small-molecule sensors that are selective for particular sugars are rare. The synthesis of BODIPYs appended with two boronic acid units is reported, alongside cellular staining/labelling and turn-on fluorescence binding data for carbohydrates. The structural frameworks were designed using computational methods, leaning on the chelation characteristics of bis(boronic acids) and the photophysical properties of BODIPYs. Selective binding to glucose is demonstrated via emission and absorption methods, and the challenges of using NMR data for studying carbohydrate binding are discussed. Furthermore, crystal structures, cell permeability and imaging properties of the BODIPYs appended with two boronic acid units are described. This work presents boronic-acid-appended BODIPYs as a potential framework for tunable carbohydrate sensing and chemical biology staining.

Direct single-molecule detection of CoA-SH and ATP by the membrane proteins TMEM120A and TMEM120B.

Membrane proteins are vital resources for developing biosensors. TMEM120A is a membrane protein associated with human pain transmission and lipid metabolism, and recent studies have demonstrated its ability to transport ions and bind to coenzyme A (COA-SH), indicating its potential to develop into a single-molecule sensor based on electrical methods. In this study, we investigated the ion transport properties of TMEM120A and its homolog TMEM120B on an artificial lipid bilayer using single-channel recording. The results demonstrate that both proteins can fuse into the lipid bilayer and generate stable ion currents under a bias voltage. Based on the stable ion transport capabilities of TMEM120A and TMEM120B, as well as the feature of TMEM120A binding with COA-SH, we developed these two proteins into a single-molecule sensor for detecting COA-SH and structurally similar molecules. We found that both COA-SH and ATP can reversibly bind to single TMEM120A and TMEM120B proteins embedded in the lipid bilayer and temporarily block ion currents during the binding process. By analyzing the current blocking signal, COA-SH and ATP can be identified at the single-molecule level. In conclusion, our work has provided two single-molecule biosensors for detecting COA-SH and ATP, offering insights for exploring and developing bio-inspired small molecule sensors.

Living Seed Materials Made by Metal–Organic Framework‐Encapsulated Bacillus Subtilis Spore

AbstractGenetically modified microorganisms (GMMs) possess significant potential for a wide range of applications; however, their execution in real‐world scenarios, particularly in environments characterized by scarce nutrients and unpredictable conditions, proves daunting. Drawing inspiration from the adaptability of natural seeds, living seed materials that combine a core–shell structure ZIF‐8 and genetically engineered spores are introduced. By genetically modifying the Bacillus subtilis strain to express distinct fluorescent proteins, the visualization of spores within the hollow sphere is facilitated, allowing for differentiation between spore and bacteria states. Importantly, the ZIF‐8 shell exhibits responsiveness to diverse nutrient levels, providing enhanced environmental resistance in comparison to bare spores upon exposure to hypochlorous acid, UV‐C radiation, and even vacuum conditions. Moreover, the potential of living seed materials for living sensors of small molecules is showcased by elucidating that spores can be resuscitated into functional bacteria after release from ZIF‐8 material. This research establishes a novel approach for the general fabrication of Metal‐Organic Frameworks (MOF)‐based living spore hybrid materials, offering environmentally controlled release of living materials across a multitude of applications.

Optical and Electrochemical Properties of a Photosensitive Pyromellitic Diimide Derivative of Cymantrene.

Photochemical properties of symmetrical pyromellitic diimide containing two cymantrenyl fragments at two nitrogen atoms were studied with IR, NMR, UV-vis, ESI-MS, and cyclic voltammetry. It was found that new unstable chelates are formed during photolysis. At the same time, the CO ligand dissociates from two Mn(CO)3 fragments during photoexcitation, which dramatically changes the electronic and redox properties of the molecule compared to the cymantrene derivative containing one imide fragment. Photolysis leads to a color change from light yellow to green. DFT calculations confirmed the possibility of the formation of complexes due to the loss of one or two CO ligands from manganese atoms. The results obtained with variation of photolysis conditions demonstrated the hemilabile character of the Mn-O=C(imide) bond. On addition of external ligands, the color and electrochemical properties changed, which is promising for the use of this complex as a sensor for small molecules.

A novel polyhedral oligomeric silsesquioxane nanohybrid fluorescent sensor designed based on an osmotic mechanism for specific detection and intelligent scavenging of magnesium ions

BackgroundMg2+ has long been recognized as one of the most vital cations due to its diverse physiological and pathological roles, making it indispensable in both biomedical and biological research. Organic fluorescent sensors are commonly employed for Mg2+ detection, but they often lack high selectivity and exhibit poor hydrophilicity, limiting their biomedical applications. ResultsHerein, we introduced a novel organic-inorganic hybrid fluorescence sensor, PFHBS, constructed on the POSS nanoplatforms. The efficient connection between PEGylated POSS and the small molecule sensor FHBS through Click chemistry enhances the selectivity and reduces interference, making this chemical sensor ideal for the accurate detection of Mg2+. Furthermore, the incorporation of POSS amplifies the ligand field effect of FHBS, making it more conducive to Mg2+ capture. The modification of PEG chains enhances the sensor's amphiphilicity, facilitating efficient cell penetration and effective Mg2+ detection at the biological level. SignificanceFinally, relying on spontaneous permeation, coupled with its strong ligand field effect and excellent cell permeability, the chemosensor demonstrates the capability to intelligently remove excess Mg2+ from the body. It has been successfully applied to mitigate renal overload resulting from acute Mg2+ poisoning.

DFT study on adsorption of small gas molecules on Pt-capped armchair and zigzag single-walled carbon nanotubes

The adsorption of small gas molecules (CO, O2 and N2) on Pt-capped armchair and zigzag single-walled carbon nanotubes (SWCNT) has been investigated by density functional theory (DFT) method to determine the potential for gas sensor detection. Results indicated that O2 and N2 molecules with lower adsorption energies (1.66 eV and 1.00 eV) exhibited weak chemical adsorption on SWCNT(4,4)-Pt. However, CO demonstrates strong chemical adsorption with a high adsorption energy of 2.23 eV on SWCNT(4,4)-Pt. The adsorption energy of CO on SWCNT(7,0)-Pt shows the chemical adsorption between CO and SWCNT(7,0)-Pt. In addition, these findings also implied Pt-capped SWCNT could act as small gas molecule sensors to detect CO toxic gas at room temperature. In this system, CO is the electron donor and SWCNT-Pt is the electron receptor. The interaction of Pt and carbon nanotube backbone delocalized π electrons could not only increases the conductivity of SWCNT-Pt, but also enhances the conductivity of SWCNT-Pt-CO. The HOMO-LUMO gap (Δε) of Pt-capped SWCNT(7,0) reduced from 0.43 eV to 0.39 eV after CO adsorption, and the Δε value of Pt-capped SWCNT(4,4) decreased by 0.08 eV. The electrical conductivity of Pt-capped armchair and zigzag SWCNT has increased after the adsorption of CO molecule. The results may be helpful for designing a promising Pt-capped SWCNT-based CO sensors.

Ratiometric Detection of Zn2+ Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permiQed BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

Nitrogen-doped carbon dots: Recent developments in its fluorescent sensor applications

Doped carbon dots have attracted great attention from researchers across disciplines because of their unique characteristics, such as their low toxicity, physiochemical stability, photostability, and outstanding biocompatibility. Nitrogen is one of the most commonly used elements for doping because of its sizeable atomic radius, strong electronegativity, abundance, and availability of electrons. This distinguishes them from other atoms and allows them to perform distinctive roles in various applications. Here, we have reviewed the most current breakthroughs in nitrogen-doped CDs (N-CDs) for fluorescent sensor applications in the last five years. The first section of the article addresses several synthetic and sustainable ways of making N-CDs. Next, we briefly reviewed the fluorescent features of N-CDs and their sensing mechanism. Furthermore, we have thoroughly reviewed their fluorescent sensor applications as sensors for cations, anions, small molecules, enzymes, antibiotics, pathogens, explosives, and pesticides. Finally, we have discussed the N-CDs' potential future as primary research and how that may be used. We hope that this study will contribute to a better understanding of the principles of N-CDs and the sensory applications that they can serve.

New generation of fluorescent probes for cell-based measurements of caspase Activation and mitochondrial superoxide

Abstract Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways. The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, with ex/em spectra of 590/610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. MitoSOX™ Green that has specificity to superoxide like MitoSOX™ Red dye. In addition, it has better spectral properties and works with GFP filters in microscopy, and allows for measurement of mitochondrial superoxide in RFP-expressing cells. MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye. These reagents are research use only, not for diagnostic purposes

Thiophene-derived “off-on-off” fluorescence chemosensors for the detection of zinc and phytate ions sequentially

In this study, thiophene-based small molecule sensors R and Q were designed and synthesized, which could monitor Zn2+ and phytate ions sequentially in aqueous solution according to “off-on-off” fluorescence. Both sensors exhibited ultrasensitive response to target ions over other reported sensors, especially toward phytate ions, with the low detection limits (3.04 × 10−10 M) and fast detection rates (<10 s). More importantly, the ethylenedioxy-modified complex Q-Zn2+ showed super selectivity for phytate ions than PPi and PO43−, without the need for pretreatment of the tested solution, which was currently an excellent fluorescence platform for the detection of phytate ions. The recognition process of target ions was explained in detail through spectral measurement and MS spectrum titration. Furthermore, the theoretical calculations were performed to provide reasonable evidence for excellent behavior of sensors. Moreover, the quantitative detection of Zn2+ and phytate ions in water samples supported strongly for application potential of both sensors.

Abstract 4781: New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide

Abstract Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways. The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, but operates in the Texas Red, 590nm excitation band, with an emission peak near 610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. Similarly, mitochondrial superoxide accompanying cell stress is probed in microscopy with the MitoSOX™ Red Mitochondrial Superoxide Indicator dye, which localizes to mitochondria and reports superoxide generation, ignoring other Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). This dye has an unusually long Stokes’ shift, requiring specialized microscopy and HCA filters that excite at 405nm, and capture emission at 610nm for specific superoxide detection. This unconventional spectroscopic profile prevents the dye’s use on many imaging platforms and promotes phototoxicity. To this end, our team has produced a dye with the same level of specificity for superoxide that will operate in one of the traditional fluorescence microscopy channels. Our candidate dye, here named MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye. These reagents are research use only, not for diagnostic purposes Citation Format: Bhaskar S. Mandavilli, Daniel Beacham, Yi Zhen Hu, Jongtae Yang, Aimei Chen. New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4781.

Zero-Background Small-Molecule Sensors for Near-IR Fluorescent Imaging of Biomacromolecular Targets in Cells.

In this study, we report a general approach to the design of a new generation of small-molecule sensors that produce a zero background but are brightly fluorescent in the near-IR spectral range upon selective interaction with a biomolecular target. We developed a fluorescence turn-on/-off mechanism based on the aggregation/deaggregation of phthalocyanine chromophores. As a proof of concept, we designed, prepared, and characterized sensors for in-cell visualization of epidermal growth factor receptor (EGFR) tyrosine kinase. We established a structure/bioavailability correlation, determined conditions for the optimal sensor uptake and imaging, and demonstrated binding specificity and applications over a wide range of treatment options involving live and fixed cells. The new approach enables high-contrast imaging and requires no in-cell chemical assembly or postexposure manipulations (i.e., washes). The general design principles demonstrated in this work can be extended toward sensors and imaging agents for other biomolecular targets.

Single-Molecule Interconversion between Chiral Configurations of Boronate Esters Observed in a Nanoreactor.

Isomers of some chemical compounds may be dynamically interconvertible. Due to a lack of sensing methods with a sufficient resolution, however, direct monitoring of such processes can be difficult. Engineered Mycobacterium smegmatis porin A (MspA) nanopores can be applied as nanoreactors so that chemical reactions can be directly monitored. Here, an MspA modified with a phenylboronic acid (PBA) adapter was prepared and was used to observe dynamic interconversion between chiral configurations of boronate esters, which appears as telegraphic switching on top of nanopore events. The mechanism of this behavior was further confirmed by trials with different halogenated catechols, dopamine, adenosine, 1,2-propanediol, and (2R,3R)-2,3-butanediol, and its generality has been demonstrated. These results suggest that an engineered MspA possesses an exceptional resolution in its monitoring of chemical reaction processes and may inspire the future design of nanopore small-molecule sensors.

Identification and characterization of RNA binding sites for (p)ppGpp using RNA-DRaCALA.

Ligand-binding RNAs (RNA aptamers) are widespread in the three domains of life, serving as sensors of metabolites and other small molecules. When aptamers are embedded within RNA transcripts as components of riboswitches, they can regulate gene expression upon binding their ligands. Previous methods for biochemical validation of computationally predicted aptamers are not well-suited for rapid screening of large numbers of RNA aptamers. Therefore, we utilized DRaCALA (Differential Radial Capillary Action of Ligand Assay), a technique designed originally to study protein-ligand interactions, to examine RNA-ligand binding, permitting rapid screening of dozens of RNA aptamer candidates concurrently. Using this method, which we call RNA-DRaCALA, we screened 30 ykkC family subtype 2a RNA aptamers that were computationally predicted to bind (p)ppGpp. Most of the aptamers bound both ppGpp and pppGpp, but some strongly favored only ppGpp or pppGpp, and some bound neither. Expansion of the number of biochemically verified sites allowed construction of more accurate secondary structure models and prediction of key features in the aptamers that distinguish a ppGpp from a pppGpp binding site. To demonstrate that the method works with other ligands, we also used RNA DRaCALA to analyze aptamer binding by thiamine pyrophosphate.

Cascade reaction-based highly sensitive fluorescent sensing systems applicable for dual-pattern fluorescence visualizing of thiophenol flavors in meat products and condiments

Thiophenols (ArSHs) are widely used as popular flavoring ingredients for making daily dishes. Dissecting the ArSHs contents in common foodstuffs is meaningful in the field of food safety science. Herein, a novel small-molecule sensor 2-(1H-benzo[d]imidazol-2-yl)-3-(2-(2,4-dinitrophenoxy)-4-morpholinophenyl)acrylonitrile (NOSA) has been tailored. The NOSA is able to respond to ArSHs, spontaneously yielding highly green-emissive fluorescent iminocoumarin (I500). This cascade reaction-based strategy is sensitive (limit-of-detection = 2.8 nM), rapid (within 5 min), and selective toward ArSH flavors. Probe NOSA has been applied to the determination of ArSHs in real-life meat products and condiments. Moreover, a far-red fluorescent compound, 2-(7-(diethylamino)-4-(4-(methylthio)styryl)-2H-chromen-2-ylidene)malononitrile (CMMT), has been first combined with NOSA to construct a composite probe NOSA@CMMT for the ratiometric detection of ArSHs (I500/I630). System NOSA@CMMT exhibits a conspicuous fluorescence change from deep-red to light-green. Benefitted from the gorgeous chromatic fluctuation, a smartphone-integrated analysis platform is established for the real-time evaluation of ArSHs level.

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools.

Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

Quantum Effects Enter Semiconductor-Based SERS: Multiresonant MoO3·xH2O Quantum Dots Enabling Direct, Sensitive SERS Detection of Small Inorganic Molecules.

There is keen research interest in building highly effective semiconductor-based surface-enhanced Raman scattering (SERS) platforms, due to their selectivity for many probe molecules and suitability for complex scenario applications. However, current tuning approaches have not yet been successful in creating semiconductor-based SERS sensors for small inorganic molecules, due to the challenge of creating sufficient SERS enhancement in semiconductors. Here, we demonstrate the use of MoO3·xH2O quantum dots (QDs), to achieve direct and sensitive fingerprinting of the inorganic species hydrazine, which is a first attempt in semiconductor-based SERS research, as well as various other probe molecules. The resulting SERS platform that uses QDs with average size of 2.2 nm could successfully detect the signal of hydrazine with a limit of detection estimated to be around 4 × 10-5 M, significantly lowering the detectable concentration by at least 1000-fold, in sharp contrast to the weak performance of 10 and 100 nm particles, demonstrating that quantum size effect triggered by small particle size below the Bohr radius is crucially responsible for high SERS activity. The significantly enhanced SERS activity is a result of vibronically coupled multipathway, highly efficient charge-transfer resonances induced by the divergence of energy states in quantum-sized MoO3·xH2O. This is a proof-of-concept demonstration of the exploitation of quantum size effect, toward significantly enhanced intrinsic SERS activity in semiconductor-based SERS materials.