Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

Low-Cost, Real-Time Polymerase Chain Reaction System for Point-of-Care Medical Diagnosis.

Global health crises due to the prevailing Coronavirus Disease 2019 (COVID-19) pandemic have placed significant strain on health care facilities such as hospitals and clinics around the world. Further, foodborne and waterborne diseases are not only spreading faster, but also appear to be emerging more rapidly than ever before and are able to circumvent conventional control measures. The Polymerase Chain Reaction (PCR) system is a well-known diagnostic tool for many applications in medical diagnostics, environmental monitoring, and food and water quality assessment. Here, we describe the design, development, and testing of a portable, low-cost, and real-time PCR system that can be used in emergency health crises and resource-poor situations. The described PCR system incorporates real-time reaction monitoring using fluorescence as an alternative to gel electrophoresis for reaction analysis, further decreasing the need of multiple reagents, reducing sample testing cost, and reducing sample analysis time. The bill of materials cost of the described system is approximately $340. The described PCR system utilizes a novel progressive selective proportional-integral-derivative controller that helps in reducing sample analysis time. In addition, the system employs a novel primer-based approach to quantify the initial target amplicon concentration, making it well-suited for food and water quality assessment. The developed PCR system performed DNA amplification at a level and speed comparable to larger and more expensive commercial table-top systems. The fluorescence detection sensitivity was also tested to be at the same level as commercially available multi-mode optical readers, thus making the PCR system an attractive solution for medical point-of-care and food and water quality assessment.

The evolution of thymic lymphomas in p53 knockout mice.

Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRß sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRß rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors' driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas.

Negative regulation of tumor suppressor p53 by microRNA miR-504.

Tumor suppressor p53 plays a central role in tumor prevention. p53 protein levels and activity are under a tight and complex regulation in cells to maintain the proper function of p53. MicroRNAs play a key role in the regulation of gene expression. Here we report the regulation of p53 through miR-504. miR-504 acts as a negative regulator of human p53 through its direct binding to two sites in the p53 3' untranslated region. Overexpression of miR-504 decreases p53 protein levels and functions in cells, including p53 transcriptional activity, p53-mediated apoptosis, and cell-cycle arrest in response to stress, and furthermore promotes tumorigenecity of cells in vivo. These results demonstrate the direct negative regulation of p53 by miR-504 as a mechanism for p53 regulation in cells, which highlights the importance of microRNAs in tumorigenesis.

Metabolic determinants in Listeria monocytogenes anaerobic listeriolysin O production.

Listeria monocytogenes is a human pathogen and a facultative anaerobe. To better understand how anaerobic growth affects L. monocytogenes pathogenesis, we first showed that anaerobic growth led to decreased growth and changes in surface morphology. Moreover, compared to aerobically grown bacteria, anaerobically grown L. monocytogenes established higher level of invasion but decreased intracellular growth and actin polymerization in cultured cells. The production of listeriolysin O (LLO) was significantly lower in anaerobic cultures-a phenotype observed in wild type and isogenic mutants lacking transcriptional regulators SigB or CodY or harboring a constitutively active PrfA. To explore potential regulatory mechanisms, we established that the addition of central carbon metabolism intermediates, such as acetate, citrate, fumarate, pyruvate, lactate, and succinate, led to an increase in LLO activity in the anaerobic culture supernatant. These results highlight the regulatory role of central carbon metabolism in L. monocytogenes pathogenesis under anaerobic conditions.

Regulation of bacterial pathogenesis by intestinal short-chain Fatty acids.

The human gut microbiota is inextricably linked to health and disease. One important function of the commensal organisms living in the intestine is to provide colonization resistance against invading enteric pathogens. Because of the complex nature of the interaction between the microbiota and its host, multiple mechanisms likely contribute to resistance. In this review, we dissect the biological role of short-chain fatty acids (SCFA), which are fermentation end products of the intestinal microbiota, in host-pathogen interactions. SCFA exert an extensive influence on host physiology through nutritional, regulatory, and immunomodulatory functions and can also affect bacterial fitness as a form of acid stress. Moreover, SCFA act as a signal for virulence gene regulation in common enteric pathogens. Taken together, these studies highlight the importance of the chemical environment where the biology of the host, the microbiota, and the pathogen intersects, which provides a basis for designing effective infection prevention and control.

Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function.

Whereas cell cycle arrest, apoptosis, and senescence are traditionally thought of as the major functions of the tumor suppressor p53, recent studies revealed two unique functions for this protein: p53 regulates cellular energy metabolism and antioxidant defense mechanisms. Here, we identify glutaminase 2 (GLS2) as a previously uncharacterized p53 target gene to mediate these two functions of the p53 protein. GLS2 encodes a mitochondrial glutaminase catalyzing the hydrolysis of glutamine to glutamate. p53 increases the GLS2 expression under both nonstressed and stressed conditions. GLS2 regulates cellular energy metabolism by increasing production of glutamate and alpha-ketoglutarate, which in turn results in enhanced mitochondrial respiration and ATP generation. Furthermore, GLS2 regulates antioxidant defense function in cells by increasing reduced glutathione (GSH) levels and decreasing ROS levels, which in turn protects cells from oxidative stress (e.g., H(2)O(2))-induced apoptosis. Consistent with these functions of GLS2, the activation of p53 increases the levels of glutamate and alpha-ketoglutarate, mitochondrial respiration rate, and GSH levels and decreases reactive oxygen species (ROS) levels in cells. Furthermore, GLS2 expression is lost or greatly decreased in hepatocellular carcinomas and the overexpression of GLS2 greatly reduced tumor cell colony formation. These results demonstrated that as a unique p53 target gene, GLS2 is a mediator of p53's role in energy metabolism and antioxidant defense, which can contribute to its role in tumor suppression.

Fatty acids regulate stress resistance and virulence factor production for Listeria monocytogenes.

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.

Regulation of female reproduction by p53 and its family members.

Tumor suppressor p53 is crucial for embryonic implantation through transcriptional up-regulation of uterine leukemia inhibitory factor (LIF). This article reports that p53 and estrogen receptor α were activated in endometrial tissues during implantation to coordinately regulate LIF production. By using human p53 knockin (Hupki) mice carrying a single nucleotide polymorphism (SNP) at codon 72 (arginine/proline), the arginine allele was demonstrated to produce higher uterine LIF levels during implantation than the proline allele. In humans, the diversity of haplotypes of the p53 gene has decreased during evolution, because the arginine allele, existing in only a subset of haplotypes, is under positive selection. This observation is consistent with previous results showing that the proline allele is enriched in patients undergoing in vitro fertilization (IVF). Studies with p63- and p73-knockout mice have demonstrated the involvement of p63 and p73 in female reproduction and their roles in egg formation and apoptosis (p63) and spindle checkpoint (p73) in female mice. Here, the role of p63 and p73 in human reproduction was investigated. Selected alleles of SNPs in p63 and p73 genes were enriched in IVF patients. These findings demonstrate that the p53 family members are involved in several steps to regulate female reproduction in mice and humans.

Single-nucleotide polymorphisms in the p53 pathway regulate fertility in humans.

The tumor suppressor protein p53 plays an important role in maternal reproduction in mice through transcriptional regulation of leukemia inhibitory factor (LIF), a cytokine crucial for blastocyst implantation. To determine whether these observations could be extended to humans, a list of single-nucleotide polymorphisms (SNPs) in the p53 pathway that can modify the function of p53 was assembled and used to study their impact on human fertility. The p53 allele encoding proline at codon 72 (P72) was found to be significantly enriched over the allele encoding arginine (R72) among in vitro fertilization (IVF) patients. The P72 allele serves as a risk factor for implantation failure. LIF levels are significantly lower in cells with the P72 allele than in cells with the R72 allele, which may contribute to the decreased implantation and fertility associated with the P72 allele. Selected alleles in SNPs in LIF, Mdm2, Mdm4, and Hausp genes, each of which regulates p53 levels in cells, are also enriched in IVF patients. Interestingly, the role of these SNPs on fertility was much reduced or absent in patients older than 35 years of age, indicating that other functions may play a more important role in infertility in older women. The association of SNPs in the p53 pathway with human fertility suggests that p53 regulates the efficiency of human reproduction. These results also provide a plausible explanation for the evolutionary positive selection of some alleles in the p53 pathway and demonstrate the alleles in the p53 pathway as a good example of antagonistic pleiotropy.

Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model.

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and upregulated expression of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease.

The Opposing Role of Propionate in Modulating Listeria monocytogenes Intracellular Infections.

Listeria monocytogenes is a Gram-positive, intracellular pathogen responsible for the highly fatal foodborne illness listeriosis. Establishing intracellular infections requires the coordinated expressions of a variety of virulence factors, such as the pore-forming toxin listeriolysin O (LLO), in response to various intra- and extracellular signals. For example, we previously reported that L. monocytogenes differentially modulated LLO production in response to exogenous propionate, a short chain fatty acid either used in salt form as a human food ingredient or produced endogenously by gut microbial fermentation. Therefore, propionate is likely a continuously present signal throughout the L. monocytogenes transmission and infection process. However, little is known about the role of propionate in modulating L. monocytogenes-host interactions. Here we investigated the impact of propionate treatment on L. monocytogenes intracellular infections using cell culture infection models. Propionate treatment was performed separately on L. monocytogenes or host cells before or during infections to better distinguish pathogen-versus-host responses to propionate. Intracellular CFU in RAW264.7 macrophages and plaque diameters in L-fibroblasts were measured as proxy for intracellular infection outcomes. Nitrite levels and cellular morphology were also measured to assess host responses to propionate. We found that propionate pretreatment of anaerobic, but not aerobic, L. monocytogenes significantly enhanced subsequent intracellular infections in both cell types and nitrite production by infected macrophages. Propionate treatment of uninfected macrophages significantly altered cell morphology, seen by longer cells and greater migration, and reduced nitrite concentration in activated macrophages. Treatment of macrophages with propionate prior to or during infections significantly inhibited intracellular growth of L. monocytogenes, including those pre-treated with propionate. These results showcased an opposing effect of propionate on L. monocytogenes intracellular infections and strongly support propionate as an important signaling molecule for both the pathogen and the host cell that can potentially alter the outcome of L. monocytogenes-host interactions.

Genetic and stochastic influences upon tumor formation and tumor types in Li-Fraumeni mouse models.

p53 is the most frequently mutated gene in human cancers. Li-Fraumeni syndrome patients inheriting heterozygous p53 mutations often have a much-increased risk to develop cancer(s) at early ages. Recent studies suggest that some individuals inherited p53 mutations do not have the early onset or high frequency of cancers. These observations suggest that other genetic, environmental, immunological, epigenetic, or stochastic factors modify the penetrance of the cancerous mutant Tp53 phenotype. To test this possibility, this study explored dominant genetic modifiers of Tp53 mutations in heterozygous mice with different genetic backgrounds. Both genetic and stochastic effects upon tumor formation were observed in these mice. The genetic background of mice carrying Tp53 mutations has a strong influence upon the tissue type of the tumor produced and the number of tumors formed in a single mouse. The onset age of a tumor is correlated with the tissue type of that tumor, although identical tumor tissue types can occur at very different ages. These observations help to explain the great diversity of cancers in different Li-Fraumeni patients over lifetimes.

Branched-chain fatty acids promote Listeria monocytogenes intracellular infection and virulence.

Anteiso-branched-chain fatty acids (BCFA) represent the dominant group of membrane fatty acids and have been established as crucial determinants in resistance against environmental stresses in Listeria monocytogenes, a facultative intracellular pathogen. Here, we investigate the role of anteiso-BCFA in L. monocytogenes virulence by using mutants deficient in branched-chain alpha-keto acid dehydrogenase (BKD), an enzyme complex involved in the synthesis of BCFA. In tissue culture models of infection, anteiso-BCFA contributed to intracellular growth and survival in macrophages and significantly enhanced plaque formation upon prolonged infection in L2 fibroblasts. The intracellular defects observed could be attributed partially to insufficient listeriolysin O (LLO) production, indicating a requirement for anteiso-BCFA in regulating virulence factor production. In a murine model of infection, the BKD-deficient mutant was highly attenuated, further emphasizing the importance of BKD-mediated metabolism in L. monocytogenes virulence. This study demonstrates an underappreciated role for BCFA in bacterial pathogenesis, which may provide insight into the development and application of antimicrobial agents.

The gut bacterial communities across six grasshopper species from a coastal tallgrass prairie.

Insect microbiomes play an important role in the health and fitness of insect hosts by contributing to nutrient absorption, immune health, and overall ecological fitness. As such, research interests in insect microbiomes have focused on agriculturally and industrially important organisms such as honey bees and termites. Orthopterans, on the other hand, have not been well explored for their resident microbial communities. Grasshoppers are an integral part of grassland ecosystems and provide important ecosystem services. Conversely, grasshoppers can be an agricultural pest requiring management with broad spectrum pesticides. However, little is known about the microbiomes of grasshoppers and their potential contribution to grasshopper biology. Here we examine the gut microbiome of six species of grasshoppers (n = 60) from a coastal tallgrass prairie ecosystem to gain a better understanding of the microbial communities present across the orthopteran order in this ecosystem. We found that there are bacterial phyla common to all six grasshopper species: Actinobacteria, Proteobacteria, Firmicutes, and to a lesser degree, Tenericutes. Although the grasshopper species shared a high relative abundance of these groups, there were notable shifts in dominant phyla depending on the grasshopper species. Moreover, measures of alpha diversity revealed a more diverse microbiome in males than females. Our observations support the hypothesis that there is a "core" group of bacterial families in these grasshopper species and factors such as trophic behaviors and the evolution of the host may contribute to the shifts in prevalence among these core microbial groups.

The Production of Listeriolysin O and Subsequent Intracellular Infections by Listeria monocytogenes Are Regulated by Exogenous Short Chain Fatty Acid Mixtures.

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

Environmental Nutrients Alter Bacterial and Fungal Gut Microbiomes in the Common Meadow Katydid, Orchelimum vulgare.

Insect gut microbiomes consist of bacteria, fungi, and viruses that can act as mutualists to influence the health and fitness of their hosts. While much has been done to increase understanding of the effects of environmental factors that drive insect ecology, there is less understanding of the effects of environmental factors on these gut microbial communities. For example, the effect of environmental nutrients on most insect gut microbiomes is poorly defined. To address this knowledge gap, we investigated the relationship between environmental nutrients and the gut microbial communities in a small study of katydids (n = 13) of the orthopteran species Orchelimum vulgare collected from a costal prairie system. We sampled O. vulgare from unfertilized plots, as well as from plots fertilized with added nitrogen and phosphorus or sodium separately and in combination. We found significantly higher Shannon diversity for the gut bacterial communities in O. vulgare from plots fertilized with added sodium as compared to those collected from plots without added sodium. In contrast, diversity was significantly lower in the gut fungal communities of grasshoppers collected from plots with added nitrogen and phosphorus, as well as those with added sodium, in comparison to those with no added nutrients. There was also a strong positive correlation between the gut bacterial and gut fungal community diversity within each sample. Indicator group analysis for added sodium plots included several taxa with known salt-tolerant bacterial and fungal representatives. Therefore, despite the small sample number, these results highlight the potential for the gut bacterial and fungal constituents to respond differently to changes in environmental nutrient levels. Future studies with a larger sample size will help identify mechanistic determinants driving these changes. Based on our findings and the potential contribution of gut microbes to insect fitness and function, consideration of abiotic factors like soil nutrients along with characteristic gut microbial groups is necessary for better understanding and conservation of this important insect herbivore.

A polymorphic variant in human MDM4 associates with accelerated age of onset of estrogen receptor negative breast cancer.

Murine double minute 4 (MDM4) shares significant structural homology with murine double minute 2 (MDM2) and interacts and regulates transcriptional activity of the tumor suppressor p53. In tumors with wild-type p53, there is often overexpression of MDM2 or MDM4 leading to functional inactivation of p53. A single-nucleotide polymorphism (SNP) in the promoter of human MDM2 (SNP309) was shown to associate with increased MDM2 expression and increased risk of cancer. This study evaluated the association of a SNP in human MDM4 (C>T) with age of onset of breast cancer in two independent cohorts. In cohort 1 of 675 patients, the average age of diagnosis for women with estrogen receptor (ER)-positive and ER-negative breast cancers was 53.2 and 48 years, respectively. In this cohort, homozygous variant (TT) carriers developed ER-negative carcinomas at an earlier age than homozygous wild-type (CC) or heterozygous (TC) such that the age at diagnosis was accelerated by 5.0 years (P = 0.018). This association was validated in a second cohort of breast cancer patients (n = 148), where TT carriers with ER-negative cancer developed the disease 3.8 years earlier than CC carriers (P = 0.006). The effect was more pronounced in Caucasians with ER-negative ductal carcinomas with TT homozygotes developing disease 7.5 years (P = 0.031) and 6.2 years (P = 7 x 10(-5)) earlier than CC carriers in cohorts 1 and 2, respectively. No association was seen in ER-positive ductal cancers suggesting that the SNP in MDM4 only has a functional association in ER-negative breast cancer.

Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors.

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.

Optothermal microbubble assisted manufacturing of nanogap-rich structures for active chemical sensing.

Guiding analytes to the sensing area is an indispensable step in a sensing system. Most of the sensing systems apply a passive sensing method, which waits for the analytes to diffuse towards the sensor. However, passive sensing methods limit the detection of analytes to a picomolar range on micro/nanosensors for a practical time scale. Therefore, active sensing methods need to be used to improve the detection limit in which the analytes are forced to concentrate on the sensors. In this article, we have demonstrated the manufacturing of nanogap-rich structures for active chemical sensing. Nanogap-rich structures are manufactured from metallic nanoparticles through an optothermally generated microbubble (OGMB) which is a laser-induced micron-sized bubble. The OGMB induces a strong convective flow that helps to deposit metallic nanoparticles to form nanogap-rich structures on a solid surface. In addition, the OGMB is used to guide and concentrate analytes towards the nanogap-rich structures for the active sensing of analytes. An active sensing method can improve the detection limit of chemical substances by an order of magnitude compared to a passive sensing method. The microbubble assisted manufacturing of nanogap-rich structures together with an active analyte sensing method paves a new way for advanced chemical and bio-sensing applications.