Recent Trends and Innovations in Bead-Based Biosensors for Cancer Detection.

Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as promising diagnostic platforms based on wide-ranging cancer biomarkers owing to the versatility, high sensitivity, and flexibility to perform the multiplexing of beads. This comprehensive review highlights recent trends and innovations in the development of bead-based biosensors for cancer-biomarker detection. We introduce various types of bead-based biosensors such as optical, electrochemical, and magnetic biosensors, along with their respective advantages and limitations. Moreover, the review summarizes the latest advancements, including fabrication techniques, signal-amplification strategies, and integration with microfluidics and nanotechnology. Additionally, the challenges and future perspectives in the field of bead-based biosensors for cancer-biomarker detection are discussed. Understanding these innovations in bead-based biosensors can greatly contribute to improvements in cancer diagnostics, thereby facilitating early detection and personalized treatments.

Caenorhabditis elegans exhibits positive gravitaxis.

BACKGROUND: Gravity plays an important role in most life forms on Earth. Yet, a complete molecular understanding of sensing and responding to gravity is lacking. While there are anatomical differences among animals, there is a remarkable conservation across phylogeny at the molecular level. Caenorhabditis elegans is suitable for gene discovery approaches that may help identify molecular mechanisms of gravity sensing. It is unknown whether C. elegans can sense the direction of gravity. RESULTS: In aqueous solutions, motile C. elegans nematodes align their swimming direction with the gravity vector direction while immobile worms do not. The worms orient downward regardless of whether they are suspended in a solution less dense (downward sedimentation) or denser (upward sedimentation) than themselves. Gravitaxis is minimally affected by the animals' gait but requires sensory cilia and dopamine neurotransmission, as well as motility; it does not require genes that function in the body touch response. CONCLUSIONS: Gravitaxis is not mediated by passive forces such as non-uniform mass distribution or hydrodynamic effects. Rather, it is mediated by active neural processes that involve sensory cilia and dopamine. C. elegans provides a genetically tractable system to study molecular and neural mechanisms of gravity sensing.

Rapid and Sensitive Pathogen Detection by DNA Amplification Using Janus Particle-Enabled Rotational Diffusometry.

Rapid and sensitive detection of infectious bacteria is in all-time high demand to prevent the further spread of the infection and allow early medical intervention. In this study, we use rotational diffusometry (RD), a natural phenomenon characterized by Janus particles, to detect pathogens like Escherichia coli by performing amplification of specific genes. This biosensing method is used to measure the change in viscosity of the fluid in the presence and absence of DNA in the solution by capturing images of modified microbeads at 10 Hz by a CCD camera followed by cross-correlation algorithm analysis. Using rotational diffusometry, we have achieved E. coli detection with 50 pg/µL DNA with a measurement time of 30 s and a sample volume of 2 µL. This sensitivity was achieved with 30 thermal cycles for three different amplicons, viz., 84, 147, and 246 bp. Meanwhile, in the case of 10 and 20 thermal cycles, the detection sensitivity was achieved with 0.1 and 1 ng/µL DNA concentrations for a 246 bp amplicon. Compared with conventional PCR, this technique appears to improve the detection time, thereby reaching a turnaround time of less than 60 min. Other studies showed a successful identification of DNA amplification up to 10 thermal cycles with different sizes of amplicons. The effect of DNA concentration, amplicon size, and the number of thermal cycles on the detection of E. coli was examined in detail and represented in the form of three maps. These maps show the clear difference and the advantages of RD method in comparison with conventional PCR. This unconventional and rapid biosensing method can be used further for downstream application of nucleic acid amplification-based pathogen detection and early disease control.

Trace Biomolecule Detection with Functionalized Janus Particles by Rotational Diffusion.

Cytokines are small proteins secreted by cells in innate and adaptive immune systems. Abnormal cytokine secretion is often regarded as an early cue of dysregulation of homeostasis due to diseases or infections. Early detection allows early medical intervention. In this study, a natural phenomenon called rotational Brownian motion was characterized by Janus particles and its potential use in detection of trace biomolecules explored. Through the functionalization of the Janus particles with an antibody, the target cytokine, that is, tumor necrosis factor-α, was measured in terms of rotational diffusion. Rotational diffusion is highly sensitive to the particle volume change according to the Stokes-Einstein-Debye relation and can be quantified by blinking signal. Accordingly, 1 µm half-gold and half-fluorescent microbeads were conjugated with 200 nm nanobeads through sandwiched immunocomplexes. The light source, lead time for stabilization, and purification were investigated for optimization. Particle images can be captured with green light at 5 Hz within 300 s. Under such conditions, the functionalized Janus particles eventually achieved a limit of detection of 1 pg/mL. The rotational diffusometry realized by Janus particles was power-free and feasible for ultrasensitive detection, such as early disease detection.

Investigation of Micro-volume Viscosity with Janus Microbeads Based on Rotational Brownian Motion.

Viscosity is an important property of liquids. A viscosity change of aqueous substances that deviates from their normal levels usually implies a compromise in quality due to degradation or microorganism proliferation. Monitoring of macro-scale viscosity can be simply realized by various conventional tools, such as rotational viscometers, capillary tubes, falling bodies, and so forth. Nevertheless, today, micro-volume viscosity measurement remains a challenging endeavor, resulting in rare, expensive, or difficult-to-obtain samples not very well studied. For this reason, a novel technique for micro-viscosity based on rotational Brownian motion is presented in this paper. Janus microbeads were made by coating fluorescent polystyrene beads with gold film. Taking advantage of the bead configuration of half gold/half fluorescence, the rotational Brownian signal was expressed in terms of blinking fluorescent intensity. The characteristic correlation time was derived from the blinking intensity of trace amounts of a selected medium over a certain time period, and results were correlated with viscosity. Given a volume of only 2 µL for each measurement, calibration of a series of glycerol⁻water mixtures (100%⁻1% (v/v) water content) yielded good agreement with the expected viscosity predictions over the range of 0.8⁻574.8 cP. Five common oil products, including lubricant oil, baby oil, food oil, olive oil, and motor oil, were further investigated to demonstrate the feasibility and practicability of the proposed technique. Data measured by the rotational Brownian motion-based diffusometer were comparable with those measured by a commercial rotational viscometer. The method also explicitly showed viscosity degradation after the oils were heated at a high temperature of over 100 °C for 10 min. Evaluation proved the proposed Janus microbead-enabled rotational diffusometric technique to be a promising approach for rapid and micro-scale viscosity measurement.

Contact-Lens Biosensors.

Rapid diagnosis and screening of diseases have become increasingly important in predictive and preventive medicine as they improve patient treatment strategies and reduce cost as well as burden on our healthcare system. In this regard, wearable devices are emerging as effective and reliable point-of-care diagnostics that can allow users to monitor their health at home. These wrist-worn, head-mounted, smart-textile, or smart-patches devices can offer valuable information on the conditions of patients as a non-invasive form of monitoring. However, they are significantly limited in monitoring physiological signals and biomechanics, and, mostly, rely on the physical attributes. Recently, developed wearable devices utilize body fluids, such as sweat, saliva, or skin interstitial fluid, and electrochemical interactions to allow continuous physiological condition and disease monitoring for users. Among them, tear fluid has been widely utilized in the investigation of ocular diseases, diabetes, and even cancers, because of its easy accessibility, lower complexity, and minimal invasiveness. By determining the concentration change of analytes within the tear fluid, it would be possible to identify disease progression and allow patient-oriented therapies. Considering the emerging trend of tear-based biosensing technology, this review article aims to focus on an overview of the tear fluid as a detection medium for certain diseases, such as ocular disorders, diabetes, and cancer. In addition, the rise and application of minimally invasive detection and monitoring via integrated contact lens biosensors will also be addressed, in regards to their practicality and current developmental progress.

Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, but the mechanism of force-specific activation of their signaling to modulate cellular function remains unclear. We have demonstrated that bone morphogenetic protein receptor (BMPR)-specific Smad1/5 can be force-specifically activated by oscillatory shear stress (OSS) in ECs to cause cell cycle progression. Smad1/5 is highly activated in ECs of atherosclerotic lesions in diseased human coronary arteries from patients with end-stage heart failure undergoing heart transplantation and from apolipoprotein E-deficient mice. Application of OSS (0.5 ± 4 dyn/cm(2)) causes the sustained activation of Smad1/5 in ECs through activations of mammalian target of rapamycin and p70S6 kinase, leading to up-regulation of cyclin A and down-regulations of p21(CIP1) and p27(KIP1) and, hence, EC cycle progression. En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the inner, but not the outer, curvature of aortic arch, nor the straight segment of thoracic aorta [corrected]. Immunohistochemical and en face examinations of the experimentally stenosed abdominal aorta in rats show high levels of phospho-Smad1/5 in ECs at poststenotic sites, where OSS occurs. These OSS activations of EC Smad1/5 in vitro and in vivo are not inhibited by the BMP-specific antagonist Noggin and, hence, are independent of BMP ligand. Transfecting ECs with Smad1/5-specific small interfering RNAs inhibits the OSS-induced EC cycle progression. Our findings demonstrate the force-specificity of the activation of Smad1/5 and its contribution to cell cycle progression in ECs induced by disturbed flow.

Immunojanus Particles for low-volume and isolation-free unlabeled characterization of small Extracellular Vesicle in biofluids: Characterization of disease type by surface marker profiling.

Small extracellular vesicles (sEVs) are vital for cellular communication and serve as critical biomarker carriers for diseases such as cancer. However, quantifying and profiling sEV surface markers presents significant challenges due to the low concentration of specific sEV-bound proteins and interference by more abundant dispersed proteins. This paper presents Immunojanus Particles (IJPs), a new method that enables the direct detection of sEVs in less than an hour without isolation. The design of IJPs incorporates fluorescent and non-fluorescent halves, utilizing rotational Brownian motion to detect captured sEVs through the change in the blinking rate, without interference from the smaller dispersed proteins. We demonstrate a detection limit of 2E5 sEVs/mL with low sample volumes and the capability to characterize sEVs directly from plasma, serum, cell culture media, and urine. In a small pilot study involving 87 subjects, including individuals with colorectal cancer, pancreatic ductal adenocarcinoma, glioblastoma, Alzheimer's disease, and healthy controls, our method accurately identified the type of disease with high 0.90-0.99 AUC in a blind setting. Compared with an orthogonal ultracentrifugation plus surface plasmon resonance (UC+SPR) method that requires about 24 hours, the sensitivity and dynamic range of IJP are better by 2 logs.

Rapid tear screening of diabetic retinopathy by a detachable surface acoustic wave enabled immunosensor.

BACKGROUND: Diabetic retinopathy (DR), a chronic and progressive microvascular complication of diabetes mellitus, substantially threatens vision and is a leading cause of blindness among working-age individuals worldwide. Traditional diagnostic methods, such as ophthalmoscopy and fluorescein angiography are nonquantitative, invasive, and time consuming. Analysis of protein biomarkers in tear fluid offers noninvasive insights into ocular and systemic health, aiding in early DR detection. This study introduces a surface acoustic wave (SAW) microchip that rapidly enhances fluorescence in bead-based immunoassays for the sensitive and noninvasive DR detection from human tear samples. RESULTS: The device facilitated particle mixing for immunoassay formation and particle concentration in the droplet, resulting in an enhanced immunofluorescence signal. This detachable SAW microchip allows the disposal of the cover glass after every use, thereby improving the reusability of the interdigital transducer and minimizing potential cross-contamination. A preliminary clinical test was conducted on a cohort of 10 volunteers, including DR patients and healthy individuals. The results demonstrated strong agreement with ELISA studies, validating the high accuracy rate of the SAW microchip. SIGNIFICANCE: This comprehensive study offers significant insights into the potential application of a novel SAW microchip for the early detection of DR in individuals with diabetes. By utilizing protein biomarkers found in tear fluid, the device facilitates noninvasive, rapid, and sensitive detection, potentially revolutionizing DR diagnostics and improving patient outcomes through timely intervention and management of this vision-threatening condition.

Detachable acoustofluidic droplet-sorter.

BACKGROUND: Cell sorting is crucial in isolating specific cell populations. It enables detailed analysis of their functions and characteristics and plays a vital role in disease diagnosis, drug discovery, and regenerative medicine. Fluorescence-activated cell sorting (FACS) is considered the gold standard for high-speed single-cell sorting. However, its high cost, complex instrumentation, and lack of portability are significant limitations. Additionally, the high pressure and electric fields used in FACS can harm cell integrity. In this work, an acoustofluidic device was developed in combination with surface acoustic wave (SAW) and droplet microfluidics to isolate single-cell droplets with high purity while maintaining high cell viability. RESULT: Human embryonic kidney cells, transfected with fluorescent reporter plasmids, were used to demonstrate the targeted droplet sorting containing single cells. The acoustofluidic sorter achieved a recovery rate of 81 % and an accuracy rate higher than 97 %. The device maintained a cell viability rate of 95 % and demonstrated repeatability over 20 consecutive trials without compromising efficiency, thus underscoring its reliability. Thermal image analysis revealed that the temperature of the interdigital transducer (IDT) during SAW operation remained within the permissible range for maintaining cell viability. SIGNIFICANCE: The findings highlighted the sensitivity and effectiveness of the developed acoustofluidic device as a tool for single-cell sorting. The detachable microfluidic chip design enables the reusability of the expensive IDT, making it cost-effective and reducing the risk of cross-contamination between different biological samples. The results underscore its capability to accurately isolate individual cells on the basis of specific criteria, showcasing its potential to advance research and clinical applications requiring precise cell sorting methodologies.

Photonic crystal enhanced immunofluorescence biosensor integrated with a lateral flow microchip: Toward rapid tear-based diabetic retinopathy screening.

Diabetic retinopathy (DR) has accounted for major loss of vision in chronic diabetes. Although clinical statistics have shown that early screening can procrastinate or improve the deterioration of the disease, the screening rate remains low worldwide because of the great inconvenience of conventional ophthalmoscopic examination. Instead, tear fluid that contains rich proteins caused by direct contact with eyeballs is an ideal substitute to monitor vision health. Herein, an immunofluorescence biosensor enhanced by a photonic crystal (PhC) is presented to handle the trace proteins suspended in the tear fluid. The PhC was constructed by self-assembled nanoparticles with a thin layer of gold coated on top of it. Then, the PC substrate was conjugated with antibodies and placed in a microchannel. When the capillary-driven tear sample flew over the PC substrate, the immunoassay enabled the formation of a sandwich antibody-antigen-antibody configuration for PhC-enhanced immunofluorescence. The use of PhC resulted in a concentration enhancement of more than tenfold compared to non-PhC, while achieving an equivalent signal intensity. The limit of detection for the target biomarker, lipocalin-1 (LCN-1), reached nearly 3 µg/ml, and the turnaround time of each detection was 15 min. Finally, a preclinical evaluation was conducted using ten tear samples. A clear trend was observed, showing that the concentrations of LCN-1 were at least twofold higher in individuals with chronic diabetes or DR than in healthy individuals. This trend was consistent with their medical conditions. The results provided a direct proof-of-concept for the proposed PhC biosensor in rapid tear-based DR screening.

Editorial: Advanced Biosensing Technologies in Medical Applications.

The unprecedented pandemic over the past three years has accelerated the developments of many cutting-edge techniques to address the challenges raised in new medical frontiers [...].

Development of a Self-Viscosity and Temperature-Compensated Technique for Highly Stable and Highly Sensitive Bead-Based Diffusometry.

Brownian motion, which is a natural phenomenon, has attracted numerous researchers and received extensive studies over the past decades. The effort contributes to the discovery of optical diffusometry, which is commonly used for micro/nano particle sizing. However, the analysis uncertainty caused by the coupling relationship among particle diameter, temperature, and fluid viscosity usually poses a barrier to precise measurement. Preventing random background noise becomes the key to achieving a high level of accuracy in diffusometry detection. Recently, Janus particles have become known as an ideal tool for resolving the rotational Brownian motion. Followed by our previous study, the rotational Brownian motion and the translational Brownian motion can be separately measured using the Janus particles. Accordingly, a simple self-viscosity and temperature-compensated technique based on the delicate removal of temperature and fluid viscosity variations through particle tracking was first proposed in this study. Consequently, the translational Brownian motion was expressed in terms of particle trajectory, whereas the rotational Brownian motion was expressed in terms of the blinking signal from the Janus particles. The algorithm was verified simulatively and experimentally in temperature (10 °C to 40 °C) and viscosity-controlled (1 mPa·s to 5 mPa·s) fields. In an evaluation of biosensing for a target protein, IFN-γ, the limit of detection of the proposed self-compensated diffusometry reached 0.45 pg/mL, whereas its uncertainties of viscosity and temperature were 96 and 15-fold lower than the pure the rotational Brownian motion counterpart, respectively. The results indicated the low-uncertainty and high-accuracy biosensing capability resulting from the self-viscosity and temperature-compensated technique. This research will provide a potential alternative to future similar bead-based immunosensing, which requires ultra-high stability and sensitivity.

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection.

Seeking optimized infectious pathogen detection tools is of primary importance to lessen the spread of infections, allowing prompt medical attention for the infected. Among nucleic-acid-based sensing techniques, loop-mediated isothermal amplification is a promising method, as it provides rapid, sensitive, and specific detection of microbial and viral pathogens and has enormous potential to transform current point-of-care molecular diagnostics. In this review, the advances in LAMP-based point-of-care diagnostics assays developed during the past few years for rapid and sensitive detection of infectious pathogens are outlined. The numerous detection methods of LAMP-based biosensors are discussed in an end-point and real-time manner with ideal examples. We also summarize the trends in LAMP-on-a-chip modalities, such as classical microfluidic, paper-based, and digital LAMP, with their merits and limitations. Finally, we provide our opinion on the future improvement of on-chip LAMP methods. This review serves as an overview of recent breakthroughs in the LAMP approach and their potential for use in the diagnosis of existing and emerging diseases.

Rotational diffusometric sensor with isothermal amplification for ultra-sensitive and rapid detection of SARS-CoV-2 nsp2 cDNA.

In the wake of a pandemic, the development of rapid, simple, and accurate molecular diagnostic tests can significantly aid in reducing the spread of infections. By combining particle imaging with molecular assays, a quick and highly sensitive biosensor can readily identify a pathogen at low concentrations. Here, we implement functionalized particle-enabled rotational diffusometry in combination with loop-mediated isothermal amplification for the rapid detection of the SARS-CoV-2 nsp2 gene in the recombinant plasmid as a proof of concept for COVID-19 diagnostics. By analyzing the images of blinking signals generated by these modified particles, the change in micro-level viscosity due to nucleic acid amplification was measured. The high sensitivity of rotational diffusometry enabled facile detection within 10 min, with a limit of detection of 70 ag/µL and a sample volume of 2 µL. Tenfold higher detection sensitivity was observed for rotational diffusometry in comparison with real-time PCR. In addition, the system stability and the effect of temperature on rotational diffusometric measurements were studied and reported. These results demonstrated the utility of a rotational diffusometric platform for the rapid and sensitive detection of SARS-CoV-2 cDNA fragments.

Facile open-well immunofluorescence enhancement with coplanar-electrodes-enabled optoelectrokinetics and magnetic particles.

Electrokinetic manipulation has been proven powerful in enhancing the sensing capability of general-purpose biochips. However, the close-form configuration of biochips and the required use of low electric conductivity limit their practicability. In this study, an open-well microfluidic system facilitated with coplanar-electrodes-enabled optoelectrokinetic concentration and magnetic particles were therefore developed to overcome these challenges. The open side achieves optoelectrokinetic manipulation for biosignal enhancement, enabling free manual operations. Magnetic particles were employed in immunoassays to facilitate the rapid onsite separation of targets. A common cytokine biomarker found in many diseases, that is, tumor necrosis factor alpha (TNF-α), was used for assessing the immunosensing system. In addition to the benefits inherited from the immunoassays, the fluorescent signal enhanced by the optoelectrokinetic technique also featured rapid enhancement in 1 min and a limit of detection of as low as 2.9 pg/mL. The open-well architecture allowed the entire immunosensing process to be completed on site without frequent off-site washing. For a practical test, the TNF-α in human tear fluids was measured by the developed device and validated with a standard enzyme-linked immunosorbent assay (ELISA). The data show consistency in terms of trend. The developed open-well optoelectrokinetic device provides an insight into future facile clinical diagnoses. By simply modifying the surface linkers on the magnetic particles, the technique can be further extended to more other trace biomarker detections.

Effects of electrotactic exercise and antioxidant EUK-134 on oxidative stress relief in Caenorhabditis elegans.

Antioxidant uptake and regular exercise are two well-acknowledged measures used for rejuvenation and oxidative stress elimination. Previous studies have revealed that moderate exercise mildly increases intracellular signaling oxidant levels and strengthens the ability of an organism to deal with escalating oxidative stress by upregulating antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase. Antioxidant supplementation directly scavenges intracellular reactive oxygen species (ROS) to reduce oxidative stress. However, research to understand the impacts of these enzymes on mitigating oxidative stress from the perspective of simple animals is limited. Herein, we show that exercise combined with antioxidant supplementation ameliorates the physiological phenotypes and markers of aging in wild-type and SOD/CAT-deficient Caenorhabditis elegans. We discovered that treated wild-type and gene-deficient worms show better survivorship, reproduction, and motility compared with their control counterparts. Assays of biochemical indices revealed that variations in sod-3 expression under different stress levels imply an inducible enzyme response resulting from exercise training and antioxidant supplementation. In addition, induced ROS resistance obtained from any type of treatment could persist for several days even after treatment cessation, thus suggesting a potential long-term antioxidative stress effect. Our findings confirm that exercise, antioxidant supplementation, and their combination could significantly improve the ability of C. elegans to withstand adverse stress. Our observations provide promising insights into future therapies of anti-oxidative stress in higher animals.

Fabrication of SiO2/PEGDA Inverse Opal Photonic Crystal with Fluorescence Enhancement Effects.

The present paper reports the fabrication of inverse opal photonic crystals (IOPCs) by using SiO2 spherical particles with a diameter of 300 nm as an opal photonic crystal template and poly(ethylene glycol) diacrylate (PEGDA) as an inverse opal material. Characteristics and fluorescence properties of the fabricated IOPCs were investigated by using the Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), reflection spectroscopy, and fluorescence microscopy. The results clearly showed that the IOPCs were formed comprising of air spheres with a diameter of ∼270 nm. The decrease in size led to a decrease in the average refractive indexes from 1.40 to 1.12, and a remarkable stopband blue shift for the IOPCs was thus achieved. In addition, the obtained results also showed a fluorescence enhancement over 7.7-fold for the Fluor® 488 dye infiltrated onto the IOPCs sample in comparison with onto the control sample.

Dynamic manipulation by light and electric fields: micrometer particles to microliter droplets.

We demonstrate a new hybrid optoelectric technique that can manipulate objects across several length scales. The technique leverages a variety of different physical mechanisms to achieve the dynamic manipulation of droplets and also the in situ concentration of colloidal particles suspended in the droplets. Various physical mechanisms such as optoelectrowetting, electrothermal flows, and ac electroosmosis are leveraged through different modes of operation of the device. Each operational mode, which is activated through the proper combination of an applied ac bias and the illumination used, is characterized by the ability to manipulate objects on a certain length scale. We also demonstrate that the device lends itself to the active control of microstructure patterns that emerge from a droplet evaporation process.

Developing Rapid Antimicrobial Susceptibility Testing for Motile/Non-Motile Bacteria Treated with Antibiotics Covering Five Bactericidal Mechanisms on the Basis of Bead-Based Optical Diffusometry.

Rapid antimicrobial susceptibility testing (AST) is an effective measure in the treatment of infections and the prevention of bacterial drug resistance. However, diverse antibiotic types and bacterial characteristics have formed complicated barriers to rapid diagnosis. To counteract these limitations, we investigated the interactions between antibiotic-treated bacteria and functionalized microbeads in optical diffusometry. The conjugation with bacteria increased the effective microbead complex size, thereby resulting in a temporal diffusivity change. The yielded data were sorted and analyzed to delineate a pattern for the prediction of antimicrobial susceptibility. The outcome showed that a completed rapid AST based on the trend of microbead diffusivity could provide results within 3 h (2 h measurement + 1 h computation). In this research, we studied four bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, and six antibiotics. Despite the different inhibitory effects caused by various antibiotics, similar trends in diffusivity alteration for all susceptible and resistant cases in the last 40 min of the 2-h measurement period were deduced. In addition, the AST results obtained using optical diffusometry showed good agreement with those acquired from the commercial instrument and conventional culture methods. Finally, we conducted a single-blinded clinical test, and the sensitivity, specificity, and accuracy of the system reached 92.9%, 91.4%, and 91.8%, respectively. Overall, the developed optical diffusometry showcased rapid AST with a small sample volume (20 µL) and low initial bacterial count (105 CFU/mL). This technique provided a promising way to achieve early therapy against microbial diseases in the future.