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Background The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area. Methods We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs. Results We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5–12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections. Conclusions We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5–12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread.

Quantitative nucleic acid amplification testing (NAAT) is a key enabling technology for infectious disease management, especially in instances where viral load informs therapeutic decisions. Inadequate access to quantitative NAATs remains a challenge to the successful deployment of antiretroviral therapy (ART) regimens for patients with chronic hepatitis B virus (CHB) in low resourced settings (LRS). Current field-deployable NAATs are generally qualitative (yes/no) rather than quantitative in nature, making them ill-suited for viral load monitoring programs for CHB patients. Here, we report the development of a proof-of-concept molecular diagnostic test, the semiquantitative ligation and amplification (SQLA) assay, which achieves semiquantitative detection of input target DNA at two independently tunable detection thresholds with a simple visual readout. The SQLA assay utilizes a duplex competitive thermophilic helicase-dependent amplification (tHDA) chemistry and can be performed in under 1 h.

Gold electrodes are some of the most prevalent electrochemical biosensor substrate materials because they are readily functionalized with thiolated biomolecules. Yet, conventional methods to fabricate gold electrodes are costly and require onerous equipment, precluding them from implementation in low-resource settings (LRS). Recently, a number of alternative gold electrode fabrication methods have been developed to simplify and lower the cost of manufacturing. These methods include screen and inkjet printing as well as physical fabrication with common materials such as wire or gold leaf. All electrodes generated with these methods have successfully been functionalized with thiolated molecules, demonstrating their suitability for use in biosensors. Here, we detail recent advances in the fabrication, characterization and functionalization of these next-generation gold electrodes, with an emphasis on comparisons between cost and complexity with traditional cleanroom fabrication. We highlight gold leaf electrodes for their potential in LRS. This class of electrodes is anticipated to be broadly applicable beyond LRS due to their numerous inherent advantages.

Background Omicron rapidly replaced delta as the predominant strain causing COVID-19 related illness in the United States (US) in December 2021, the same month the US CDC reduced the recommended isolation period from 10 to 5 days for asymptomatic individuals or those with resolving symptoms. New evidence suggests some asymptomatic individuals with omicron remain culture positive beyond 5 days from diagnosis. We sought to evaluate the performance of a SARS-CoV-2 antigen rapid diagnostic test (RDT) in predicting persistent potential for transmission at the end of a five-day isolation period among young, fully vaccinated individuals in a university community setting. Methods A subgroup of participants enrolled in a longitudinal COVID-19 cohort were asked to self-perform RDTs on days 4 to 6 from diagnostic test date in addition to a separate self-collected anterior nasal swab used for culture and RT-PCR, and a daily symptom screen (15 COVID-19 symptom questions on a 4-point scale). We calculated the daily and overall sensitivity and specificity of the RDTs in comparison to SARS-CoV-2 culture result. We also compared the N1 cycle threshold (CT) values and symptom score on each day of the study by RDT results. Results Of 23 participants, the mean age was 20 years, all had completed their primary COVID-19 vaccine series, and 13 (65.0%) had received a booster vaccine (Table 1). Compared to culture, sensitivity and specificity of the RDTs were 100% and 62% respectively (Table 2). Compared to participants with negative RDTs, median CT values were lower in those with positive RDTs on each day of the study (Figure 1). Participants who had positive RDTs on all three days had higher symptom scores (Figure 2) than those without. Conclusion RDTs have a high sensitivity in detecting culture positive SARS-CoV-2 on Days 4 to 6 from initial diagnostic test. However, the high false positive rate of 38% means that over a third of culture negative individuals will stay in isolation longer than necessary if RDTs are used in test to release from isolation protocols. Viral loads (CT values) and symptom scores were higher for participants with persistently positive RDT result. An approach that uses a combination of RDTs, CT values and symptom score may prove useful in guiding isolation duration. Disclosures Davidson H. Hamer, MD, Trinity Biotech: Advisor/Consultant Catherine M. Klapperich, Ph.D., BioSens8, LLC: Ownership Interest.

Electrochemical biosensors are promising technologies for detection and monitoring in low-resource settings due to their potential for easy use and low-cost instrumentation. Disposable gold screen-printed electrodes (SPEs) are popular substrates for these biosensors, but necessary dopants in the ink used for their production can interfere with biosensor function and contribute to the heterogeneity of these electrodes. We recently reported an alternative disposable gold electrode made from gold leaf generated using low-cost, equipment-free fabrication. We have directly compared the surface topology, biorecognition element deposition, and functional performance of three disposable gold electrodes: our gold leaf electrodes and two commercial SPEs. Our leaf electrodes significantly outperformed the SPEs for reproducible and effective biosensing in a DNase I assay and are nearly an order of magnitude less expensive than the SPEs. Therefore, these electrodes are promising for further development as point-of-care diagnostics, especially in low-resource settings.

Disease prevalence is highest in low-resource settings (LRS) due to the lack of funds, infrastructure, and personnel required to carry out laboratory-based molecular tests. In high-resource settings, gold-standard molecular tests for diseases consist of nucleic acid amplification tests (NAATs) due to their excellent sensitivity and specificity. These tests require the extraction, amplification, and detection of nucleic acids from clinical samples. In high-resource settings, all three of these steps require highly specialized, costly, and onerous equipment that cannot be used in LRS. Nucleic acid extraction involves multiple centrifugation steps. Amplification consists of the polymerase chain reaction (PCR), which requires thermal cyclers. The detection of amplified DNA is typically done with specialized thermal cyclers that are capable of fluorescence detection. Traditional methods used to extract, amplify, and detect nucleic acids cannot be used outside of a laboratory in LRS. Thus, there is a need for affordable point-of-care devices to ease the high burden of disease in LRS.The past decade of work on paper-based fluidic devices has resulted in the invention of many paper-based biosensors for disease detection as well as isothermal amplification techniques that replace PCR. However, a challenge still remains in detecting pathogenic biomarkers from complex human samples without specialized laboratory equipment. Our research has focused on the development of affordable technologies to extract and detect nucleic acids in clinical samples with minimal equipment. Here we describe methods for the paper-based extraction, amplification, and detection of nucleic acids. This Account provides an overview of our latest technologies developed to detect an array of diseases in low-resource settings. We focus on detecting nucleic acids of H1N1, human papillomavirus (HPV), Neisseria gonorrheae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), and malaria from a variety of clinical sample types. H1N1 RNA was extracted from nasopharyngeal swabs; HPV, NG, and CT DNA were extracted from either cervical, urethral, or vaginal swabs; TV DNA was extracted from urine; and malaria DNA was extracted from whole blood. Different sample types necessitate different nucleic extraction protocols; we provide guidelines for assay design based on the clinical sample type used. We compare the pros and cons of different isothermal amplification techniques, namely, helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), and a novel isothermal amplification technique that we developed: isothermal-identical multirepeat sequences (iso-IMRS). Finally, we compare various detection mechanisms, including lateral-flow and electrochemical readouts. Electrochemical readouts frequently employ gold electrodes due to strong gold-thiol coupling. However, the high cost of gold precludes their use in LRS. We discuss our development of novel gold leaf electrodes that can be made without specialized equipment for a fraction of the cost of commercially available gold electrodes.

We describe an electrochemical strategy to transduce allosteric transcription factor (aTF) binding affinity to sense steroid hormones. Our approach utilizes square wave voltammetry (SWV) to monitor changes in current output as a progesterone (PRG) specific aTF (SRTF1) unbinds from the cognate DNA sequence in the presence of PRG. The sensor detects PRG in artificial urine samples with sufficient sensitivity suitable for clinical applications. Our results highlight the capability of using aTFs as the biorecognition elements to develop electrochemical point-of-care biosensors for detection of small molecule biomarkers and analytes.

Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (

International audience; Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here we present the development and assessment of an immobilized quantum dot - transcription factor - nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor is composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, and embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot - transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot - transcription factor DNA assembly resulting in an attenuated ratiometric fluorescent output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeat analyte measurements are also similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor - DNA binding as a recognition/ transduction element, offering a promising framework for future biosensor devices based upon allosteric transcription factor.

Loop-mediated amplification (LAMP) is an isothermal amplification technique favored in diagnostics and point-of-care work due to its high sensitivity and ability to run in isothermal conditions. In addition, a visual readout by lateral flow strips (LFS) can be used in conjunction with LAMP, making the assay accessible at the point-of-care. However, the amplicons resulting from a LAMP reaction varied in length and shape, making them undiscernible on a double-stranded DNA intercalating dye stained gel. Standard characterization techniques also do not identify which amplicons specifically bind to the LFS, which generate the visual readout. We aimed to standardize our characterization of LAMP products during assay development by using fluorescein amidite (FAM) and biotin-tagged loop forward and backward primers during assay development. A pvuII restriction enzyme digest is applied to the LAMP products. FAM-tagged bands are directly correlated with the LFS visual readout. We applied this assay development workflow for an HPV 16 assay using both plasmid DNA and clinical samples to demonstrate proof of concept for generalized assay development work. Electronic supplementary material The online version of this article (10.1007/s00216-020-02702-9) contains supplementary material, which is available to authorized users.

Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices., Bacteria represent an unexploited reservoir of biosensing proteins. Here the authors use genomic screens and functional assays to isolate a progesterone sensing allosteric transcription factor and use a FRET-based method to develop an optical progesterone sensor.

Estimated to be the most common non-viral sexually transmitted infection globally, Trichomonas vaginalis (TV) can lead to pelvic inflammatory disease, pregnancy complications, and increased risk of acquiring and transmitting HIV. Once diagnosed, TV infection can be treated with oral antibiotics; however, infected individuals are often asymptomatic and do not seek treatment. The WHO and others have identified a need for point-of-care tests to expand access to TV testing and screening; ideal test characteristics include high sensitivity and specificity and the ability to use urine as a sample type, rather than invasively collected swab samples. Here, we report on a proof-of-concept prototype for rapid, electrostatic enrichment of DNA from urine samples and demonstrate the use of large volumes of urine to increase sensitivity of downstream nucleic acid amplification testing. We developed an internally controlled thermophilic helicase-dependent amplification (tHDA) assay with lateral flow immunoassay readout and demonstrate that this tHDA assay can be performed directly on our DNA capture filter. We validated our method using clinical urine samples with qPCR-quantified TV loads. Using 62 clinical urine samples and a simple sample processing device, our tHDA assay displayed 96.6% sensitivity and 100% specificity. Our analytical limit of detection was found to be approximately 7 genomic equivalents of TV DNA per mL of sample when 1 mL of sample was tested, comparable to existing isothermal tests for TV. Using large-volume simulated samples (40 mL of buffered urine with spiked-in TV DNA), we also demonstrated that sensitivity could be improved 28-fold to 0.25 genomic equivalents of TV DNA per mL, with a sample processing time of only 2 minutes.

The COVID-19 pandemic has increased the use of rapid antigen tests such as the Abbott BinaxNOWTM COVID-19 Antigen Self-Test. In winter of 2021-2022, the omicron variant surge made it quickly apparent that although rapid diagnostic tests (RDTs) are less sensitive than qRT-PCR, the accessibility, ease of use, and rapid read-outs of RDTs made them a sought after and often sold-out item at local suppliers. Here, we sought to qualify the BinaxNOWTM test for use in our university testing program as a method to rule-in positive or rule-out negative individuals quickly when they seek care at our priority qRT-PCR testing site. To perform this qualification study, we collected matched additional swabs from individuals attending this test site for standard of care qRT-PCR testing. All matched swabs were tested using the BinaxNOWTM RDT. Initially as part of a feasibility study, test period 1 (n=110) samples were put in cold storage prior to testing. In follow-on test period (n=209), we tested samples real-time at the test facility. Combined, 102 of 319 samples tested positive for SARS-CoV-2. All samples for which genome sequence could be collected were omicron (n=92). We observed a calculated sensitivity of 53.9%, specificity of 100%, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 82.2% for the BinaxNOWTM tests (n=319). Sensitivity improved (75.3%) by changing the qRT-PCR positivity threshold from a CT of 40 to a CT of 30. The ROC curve shows that for qRT-PCR positive CT values between 24-40, the BinaxNOWTM test is of limited value diagnostically. Our results suggest that RDT tests could be used in our setting to confirm SARS-CoV-2 infection in individuals with substantial viral load, but that a significant fraction of infected individuals would be missed if we used RDT tests exclusively to rule out infection.

The Omicron variant of SARS-CoV-2 is highly transmissible in vaccinated and unvaccinated populations. The dynamics governing its establishment and propensity towards fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. In this work, we describe the dynamics of Omicron at three institutions of higher education (IHEs) in the greater Boston area.We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction to three IHEs with asymptomatic surveillance programs.We show that the establishment of Omicron at IHEs precedes that of the state and region, and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2-3 day delay. Finally, we compare cycle threshold (Ct) values in Omicron vs. Delta variant cases on college campuses, and identify lower viral loads among college affiliates harboring Omicron infections.We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread.

RT-qPCR has been used as the gold standard method for detecting SARS-CoV-2 since early in the pandemic. At our university based high throughput screening program, we test all members of our community weekly. RT-qPCR cycle threshold (CT) values are inversely proportional to the amount of viral RNA in a sample, and thus are a proxy for viral load. We hypothesized that CT values would be higher, and thus the viral loads at the time of diagnosis would be lower in individuals who were infected with the virus but remained asymptomatic throughout the course of the infection. We collected the N1 and N2 CT values from 1633 SARS-CoV-2 positive RT-qPCR tests of individuals sampled between August 7, 2020, and March 18, 2021, at the BU Clinical Testing Laboratory. We matched this data with symptom reporting data from our clinical team. We found that asymptomatic patients had CT values significantly higher than symptomatic individuals on the day of diagnosis. Symptoms were followed by the clinical team for 10 days post the first positive test. Within the entire population, 78.1% experienced at least one symptom during surveillance by the clinical team (n=1276/1633). Of those experiencing symptoms, the most common symptoms were nasal congestion (73%, n=932, 1276), cough (60.0%, n=761/1276), fatigue (59.0%, n=753/1276), and sore throat (53.1%, n=678/1276). The least common symptoms were diarrhea (12.5%, n=160/1276), dyspnea on exertion (DOE) (6.9%, n=88/1276), foot or skin changes (including rash) (4.2%, n=53/1276), and vomiting (2.1%, n= 27/1276). Presymptomatic individuals, those who were not symptomatic on the day of diagnosis but became symptomatic over the following 10 days, had CT values higher for both N1 (median= 27.1, IQR 20.2- 32.9) and N2 (median=26.6, IQR 20.1-32.8) than the symptomatic group N1 (median= 21.8, IQR 17.2- 29.4) and N2 (median= 21.4, IQR 17.3- 28.9) but lower than the asymptomatic group N1 (median=29.9, IQR 23.6-35.5) and N2 (median= 30.0, IQR 23.1- 35.7). This study supports the hypothesis that viral load in the anterior nares on the day of diagnosis is a measure of disease intensity at that time.

In 2019, the first cases of SARS-CoV-2 were detected in Wuhan, China, and by early 2020 the cases were identified in the United States. SARS-CoV-2 infections increased in the US causing many states to implement stay-at-home orders and additional safety precautions to mitigate potential outbreaks. As policies changed throughout the pandemic and restrictions lifted, there was an increase in demand for Covid-19 testing which was costly, difficult to obtain, or had long turn-around times. Some academic institutions, including Boston University, created an on-campus Covid-19 screening protocol as part of planning for the safe return of students, faculty, and staff to campus with the option for in-person classes. At BU, we stood up an automated high-throughput clinical testing lab with the capacity to run 45,000 individual tests weekly by fall of 2020, with a purpose-built clinical testing laboratory, a multiplexed RT-PCR test, robotic instrumentation, and trained CLIA certified staff. There were challenges to overcome, including the supply chain issues for PPE testing materials, and equipment that were in high demand. The Boston University Clinical Testing Laboratory was operational at the start of the fall 2020 academic year. The lab performed over 1 million SARS-CoV-2 RT-PCR tests during the 2020-2021 academic year.

Key Points Question Can a multifaceted approach lead to control of COVID-19 transmission and spread on an urban campus? Findings In this case series including more than 500 000 COVID-19 tests and 719 individuals with COVID-19 at Boston University, active surveillance of campus populations, isolation of individuals with SARS-CoV-2 infection, early and vigorous contact tracing and quarantine, regular communication, robust data systems, and strong leadership were associated with minimal transmission of SARS-CoV-2. Most transmission occurred off campus, and there was no evidence of classroom transmission. Meaning These findings suggest that using frequent testing, vigorous contact tracing, rapid isolation and quarantine, and a strong leadership structure to ensure rapid decision-making and adaption to emerging data, controlling the spread of SARS-CoV-2 on an urban campus was feasible despite worsening local transmission during the semester., This case series describes the use of a multifaceted COVID-19 control plan to reduce spread of SARS-CoV-2 at a large urban university during the second wave of the pandemic., Importance The COVID-19 pandemic has severely disrupted US educational institutions. Given potential adverse financial and psychosocial effects of campus closures, many institutions developed strategies to reopen campuses in the fall 2020 semester despite the ongoing threat of COVID-19. However, many institutions opted to have limited campus reopening to minimize potential risk of spread of SARS-CoV-2. Objective To analyze how Boston University (BU) fully reopened its campus in the fall of 2020 and controlled COVID-19 transmission despite worsening transmission in Boston, Massachusetts. Design, Setting, and Participants This multifaceted intervention case series was conducted at a large urban university campus in Boston, Massachusetts, during the fall 2020 semester. The BU response included a high-throughput SARS-CoV-2 polymerase chain reaction testing facility with capacity to deliver results in less than 24 hours; routine asymptomatic screening for COVID-19; daily health attestations; adherence monitoring and feedback; robust contact tracing, quarantine, and isolation in on-campus facilities; face mask use; enhanced hand hygiene; social distancing recommendations; dedensification of classrooms and public places; and enhancement of all building air systems. Data were analyzed from December 20, 2020, to January 31, 2021. Main Outcomes and Measures SARS-CoV-2 diagnosis confirmed by reverse transcription–polymerase chain reaction of anterior nares specimens and sources of transmission, as determined through contact tracing. Results Between August and December 2020, BU conducted more than 500 000 COVID-19 tests and identified 719 individuals with COVID-19, including 496 students (69.0%), 11 faculty (1.5%), and 212 staff (29.5%). Overall, 718 individuals, or 1.8% of the BU community, had test results positive for SARS-CoV-2. Of 837 close contacts traced, 86 individuals (10.3%) had test results positive for COVID-19. BU contact tracers identified a source of transmission for 370 individuals (51.5%), with 206 individuals (55.7%) identifying a non-BU source. Among 5 faculty and 84 staff with SARS-CoV-2 with a known source of infection, most reported a transmission source outside of BU (all 5 faculty members [100%] and 67 staff members [79.8%]). A BU source was identified by 108 of 183 undergraduate students with SARS-CoV-2 (59.0%) and 39 of 98 graduate students with SARS-CoV-2 (39.8%); notably, no transmission was traced to a classroom setting. Conclusions and Relevance In this case series of COVID-19 transmission, BU used a coordinated strategy of testing, contact tracing, isolation, and quarantine, with robust management and oversight, to control COVID-19 transmission in an urban university setting.

The present state of reproductive and sexual health around the world reveals disparities in contraceptive use and effectiveness. Unintended pregnancy and sexually transmitted infection (STI) transmission rates remain high even with current prevention methods. The 20(th) century saw a contraceptive revolution with biomedical innovation driving the success of new contraceptive technologies with central design concepts and materials. Current modalities can be broadly categorized according to their mode of function: reversible methods such as physical/chemical barriers or hormonal delivery devices via systemic (transdermal and subcutaneous) or localized (intrauterine and intravaginal) administration, and nonreversible sterilization procedures such as tubal ligation and vasectomy. Contraceptive biomaterials are at present dominated by well-characterized elastomers such as polydimethylsiloxane and ethylene vinyl acetate due to their favorable material properties and versatility. Contraceptives alter the normal function of cellular components in the reproductive systems to impair fertility. The purpose of this review is to highlight the bioengineering design of existing methods, explore novel adaptations, and address notable shortcomings in current contraceptive technologies.

Developing ultrasensitive methods capable of detecting submicroscopic parasitemia-a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence-is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity but are limited by the number of copies of the amplification targets. Herein, we perform a novel genome mining approach to identify a pair of identical multirepeat sequences (IMRSs) that constitute 170 and 123 copies in the Plasmodium falciparum genome and explore their potential as primers for PCR. Real-time quantitative PCR analyses have shown the ability of P. falciparum IMRSs to amplify as low as 2.54 fg of P. falciparum genomic DNA (approximately 0.1 parasite), with a striking 100-fold increase in detection limit when compared with P. falciparum 18S rRNA (251.4 fg; approximately 10 parasites). Validation with clinical samples from malaria-endemic regions has shown 6.70 ± 1.66 cycle better detection threshold in terms of Ct value for P. falciparum IMRSs, with approximately 100% sensitivity and specificity. Plasmodium falciparum IMRS assays are also capable of detecting submicroscopic infections in asymptomatic samples. To summarize, this approach of initiating amplification at multiple loci across the genome and generating more products with increased analytical sensitivity is different from classic approaches amplifying multicopy genes or tandem repeats. This can serve as a platform technology to develop advanced diagnostics for various pathogens.

Asymptomatic surveillance testing together with COVID-19-related research can lead to positive SARS-CoV-2 tests resulting not from true infections, but non-infectious, non-hazardous by-products of research (amplicons). Amplicons can be widespread and persistent in lab environments and can be difficult to distinguish for true infections. We discuss prevention and mitigation strategies., Asymptomatic surveillance testing is a powerful approach for rapid detection of SARS-CoV-2, currently utilized by multiple research institutes as part of routine pandemic response protocols. In combination with the recent explosion in COVID-19 related research, many research labs are working with SARS-CoV-2 genetic material. This raises a critical issue: some positive test results are due to the detection of the non-infectious, non-hazardous genetic sequences used for research (amplicons) instead of true COVID-19 infections. By collecting positive test results from several of the universities associated with this study, we identified a cohort of individuals working with SARS-CoV-2 genetic material for non-diagnostic purposes. Follow-up PCR and antibody tests on a subset of 39 individuals showed that 36 of those were not true COVID-19 cases, but instead their positive test result likely arose from amplicon contamination. In research labs working with genetic materials derived from the virus, environmental testing indicated amplicon residue might be present on surfaces including on notebooks, keyboards, drinking glasses, and doorknobs. Removal of individuals from standard testing protocols, per CDC guidelines for positive cases, risks the spread of true infection if the individuals were to be truly infected during the recommended 90 days no-testing period after an initial positive test. On the basis of this risk, combined with unnecessary isolation, quarantine, and contact tracing, we argue that it is critical to minimize instances of amplicon contamination and develop protocols for handling suspected cases to propel research efforts and avoid diverting resources from patients with COVID-19. Although our results show how amplicon contamination in research environments can lead to false positives, they also demonstrate that it is important to perform follow-up tests of those asymptomatic individuals because some might be cases of true infection. Given that amplicon contamination has not been a problem in CLIA labs, we discuss potential prevention and mitigation strategies and emphasize the relevance of this study beyond this pandemic.

ImportanceThe coronavirus disease 2019 (COVID-19) pandemic has severely disrupted United States educational institutions. Given potential adverse financial psychosocial effects of campus closures, many institutions developed strategies to reopen campuses in the fall despite the ongoing threat of COVID-19. Many however opted to have limited campus re-opening in order to minimize potential risk of spread of SARS-CoV-2.ObjectiveTo analyze how Boston University (BU) fully reopened its campus in the fall of 2020 and controlled COVID-19 transmission despite worsening transmission in the city of Boston.DesignMulti-faceted intervention case study.SettingLarge urban university campus.InterventionsThe BU response included a high-throughput SARS-CoV-2 PCR testing facility with capacity to delivery results in less than 24 hours; routine asymptomatic screening for COVID-19; daily health attestations; compliance monitoring and feedback; robust contact tracing, quarantine and isolation in on campus facilities; face mask use; enhanced hand hygiene; social distancing recommendations; de-densification of classrooms and public places; and enhancement of all building air systems.Main Outcomes and MeasuresBetween August and December 2020, BU conducted >500,000 COVID-19 tests and identified 719 individuals with COVID-19: 496 (69.0%) students, 11 (1.5%) faculty, and 212 (29.5%) staff. Overall, about 1.8% of the BU community tested positive. Of 837 close contacts traced, 86 (10.3%) tested positive for COVID-19. BU contact tracers identified a source of transmission for 51.5% of cases with 55.7% identifying a source outside of BU. Among infected faculty and staff with a known source of infection, the majority reported a transmission source outside of BU (100% for faculty and 79.8% for staff). A BU source was identified by 59.2% of undergraduate students and 39.8% of graduate students; notably no transmission was traced to a classroom setting.Conclusions and RelevanceBU was successful in containing COVID-19 transmission on campus while minimizing off campus acquisition of COVID-19 from the greater Boston area. A coordinated strategy of testing, contact tracing, isolation and quarantine, with robust management and oversight, can control COVID-19 transmission, even in an urban university setting.Key PointsQuestionCan a multi-faceted approach lead to control of COVID-19 transmission and spread on an urban campus?FindingsDespite a second wave of SARS-CoV-2 in the greater Boston area, Boston University was able to minimize outbreaks by means of active surveillance of campus populations, isolation of infected individuals, early, effective contact tracing and quarantine, regular communication, excellent data systems, and strong leadership. Most transmission appeared to occur off campus and there was no evidence of classroom transmission.MeaningUsing the main axioms of infection control including frequent testing, vigorous contact tracing, and rapid isolation and quarantine, and a strong leadership structure to ensure nimble decision-making and rapid adaption to emerging data, controlling the transmission and spread of SARS-CoV-2 on an urban campus was feasible despite worsening local transmission during the course of the semester.

Monitoring the adherence of patients taking highly active antiretroviral therapy (HAART) is a key step in treating an HIV infection, especially in resource-limited settings in the developing world. For most regimens, when patients are not at least 95% adherent to their drug schedule, there is a loss of effectiveness in treatment resulting in increases in health care costs, increases in the rate of transmission, and reduction of positive patient outcomes. Currently, subjective methods such as pill counting, electronic drug monitoring, and patient self-reporting are the only ways clinicians can track adherence and intervene in cases of noncompliance. We address this issue by developing a dipstick-based point-of-care azide-alkyne click chemistry assay with colorimetric read-out that directly tests for the presence of one common HAART drug in patient urine. An alkyne-modified dextran was synthesized and characterized by NMR and then used to colorimetrically report the presence of azidothymidine, an azide-containing HAART drug, in urine samples. The assay is specific to azide-containing molecules that are not naturally present in the urine and is sensitive to physiologically relevant urine concentrations as low as 750 uM. This point-of-care device is a strong alternative in resource-limited settings over current direct measurement techniques that are expensive and require trained users such as HPLC.

Significance Detection of submicroscopic malaria in asymptomatic individuals is needed for eradication and remains a diagnostic gap in resource-limited settings. Nonfalciparum clinical diagnostics are a second gap, as these infections have a low parasite density and are commonly undetected. We describe an integrated, 60-min, ultrasensitive and specific CRISPR-based diagnostic for the four major pathogenic Plasmodium species that can fill these gaps. Using the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform, we designed assays with limits of detection below that recommended by the World Health Organization. These assays have a simplified sample preparation method: the SHERLOCK parasite rapid extraction protocol, which eliminates complicated nucleic acid extraction steps. Our work further translates the SHERLOCK platform into a field-deployable diagnostic., Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (

International audience; Immobilization of biosensors on surfaces is a key step toward development of devices for real‐world applications. Here the preparation, characterization, and evaluation of a surface‐bound transcription factor–nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore‐labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot (QD). Upon addition of anhydrotetracycline (aTc)—the analyte—the TetR‐QDs release from the surface‐bound DNA, resulting in loss of the Förster resonance energy transfer signal. The sensor responds in a dose‐dependent manner over the relevant range of 0–200 µm aTc with a limit of detection of 80 nm . The fabrication of the sensor and the subsequent real‐time quantitative measurements establish a framework for the design of future surface‐bound, affinity‐based biosensors using allosteric transcription factors for molecular recognition.

Nucleic acid amplification tests (NAATs), which amplify and detect pathogen nucleic acids, are vital methods to diagnose diseases, particularly in cases where patients exhibit low levels of infection. For many blood-borne pathogens such as HIV or Plasmodium, it is necessary to first extract pathogen RNA or DNA from patient blood prior to analysis with NAATs. Traditional nucleic acid extraction methods are expensive, resource-intensive and are often difficult to deploy to resource-limited areas where many blood-borne infections are widespread. Here, we describe a portable, paper-and-plastic device for instrument-free nucleic acid extraction from whole blood, which we call SNAPflex, that builds upon our previous work extracting RNA in a 2D platform from nasopharyngeal swabs. We demonstrated improved extraction of HIV RNA from simulated patient samples compared to traditional extraction methods and long-term stability of extracted RNA without the need for cold storage. We further demonstrated successful extraction and recovery of Plasmodium falciparum DNA from simulated patient samples with superior recovery compared to existing extraction methods. The SNAPflex device extracts and purifies DNA and RNA from whole blood which can be amplified with traditional NAATs, and was designed to easily manufacture and integrate into existing health systems.

Malaria continues to be one of the most devastating diseases impacting global health. Although there have been significant reductions in global malaria incidence and mortality rates over the past 17 years, the disease remains endemic throughout the world, especially in low- and middle-income countries. The World Health Organization has put forth ambitious milestones moving toward a world free of malaria as part of the United Nations Millennium Goals. Mass screening and treatment of symptomatic and asymptomatic malaria infections in endemic regions is integral to these goals and requires diagnostics that are both sensitive and affordable. Lab-on-a-chip technologies provide a path toward sensitive, portable, and affordable diagnostic platforms. Here, we review and compare currently-available and emerging lab-on-a-chip diagnostic approaches in three categories: (1) protein-based tests, (2) nucleic acid tests, and (3) cell-based detection. For each category, we highlight the opportunities and challenges in diagnostics development for malaria elimination, and comment on their applicability to different phases of elimination strategies.

Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

The glucose biosensor, built upon the redox enzyme glucose oxidase, is the most commercially successful and studied enzymatic biosensor. However, the lack of available and functionally validated enzymes is prohibiting the development of redox-based sensors for other important analytes. Herein, we present the development and assessment of an electrochemical nicotine biosensor, using genomic screening to identify the gene for a known nicotine catabolizing redox enzyme. The resulting nicotine biosensor demonstrated a specific, sensitive, and stable operational profile with a limit of detection of 27 μM over the range of 0-200 μM. This range is well within the physiological concentrations of nicotine present in smoker urine. Specificity and cross-reactivity were measured against structurally similar compounds to nicotine as well as to known physiological by-products. Our results highlight that this novel enzymatic electrochemical nicotine biosensor possesses operational capabilities for monitoring of nicotine in physiologically relevant conditions. The screening methodology can be generalized for the discovery of enzymes for novel sensor development.

There is an immense potential for using biosensors in medical diagnostics as well as industries like pharmaceutical, food, beverages, environmental, and agricultural. Today, there remains a significant challenge to meet low levels of detection without compromising simplicity and affordability. Hormone measurements play a central role in family planning programs benefiting millions of women, and in monitoring endocrine disorders. Progesterone is a steroid hormone secreted by the corpus luteum and tracking progesterone levels during an ovulation cycle is beneficial for increasing fertility odds. Furthermore, during pregnancy, progesterone balance helps nurture and develop the fetus. Currently, there are no convenient and cost effective point-of-care (POC) devices to detect progesterone: antibody based approaches are used for progesterone sensing but their use is limited by high cost, production duration, and unreliability. This work has led to the development of in vitro biosensors based on bacterial allosteric transcription factors (aTFs). Like antibodies, aTFs recognize analytes with high sensitivity and specificity. In contrast to antibodies, aTFs respond to analyte binding by changing their affinity for a cognate DNA sequence. Using a novel progesterone-specific aTF, and its binding affinity to a specific DNA sequence provides an intrinsic electrochemical transduction mechanism for the development of novel progesterone biosensor. An electrochemical transduction method offers higher sensitivity and specificity at low cost. The biosensor surface includes a gold electrode with a surface immobilized DNA-aTF complex. During electrochemical measurements using a negative redox mediator (ferrocyanide/ferricyanide complex), in the absence of progesterone the overall negative charge of DNA-aTF complex blocks the negative mediator, prevents exchange of electrons with gold electrode, and generates lower current output. Addition of analyte or progesterone alters the affinity binding of DNA-aTF, releases the aTF from the surface, and enables the mediator to reach the surface, thereby resulting in an amplified current output. Preliminary results using our biosensor yielded a limit of detection of 33 nM over a physiologically relevant range of 0-10 µM progesterone in buffer, and 44 nM in presence of interferents in artificial urine.

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.

Proper management of an HIV infection requires that a patient be at least 80–95% adherent to a prescribed drug regimen to avoid poor health outcomes and the development of drug-resistant HIV strains. Clinicians generally monitor adherence habits indirectly through patient self-reporting, pill counting, and electronic drug monitoring. While direct measurement of patient samples like urine for monitoring drug levels is possible, it requires specialized equipment and training that is not readily available in resource-limited settings where the need is greatest. In this work we report the development of an antibody that binds to tenofovir (TFV), a key small molecule drug for both the treatment and prevention of HIV, and a competitive lateral flow assay that uses that antibody to monitor urine samples for the presence of the drug. TFV was conjugated to an immunogenic protein and injected into rabbits to raise polyclonal antibodies sensitive to the drug. The antibodies were verified for TFV-sensitivity by immunoprecipitation and HPLC. A gold nanoparticle-based competitive assay was developed to detect the presence of TFV in urine samples with a sensitivity of 1 μg mL−1. This TFV assay could be deployed as a point-of-care device for adherence monitoring in resource-limited settings as a low-cost, accurate, and speedy alternative to current methods to better inform changes in treatment.

We report the first demonstration of using heat on a paper device to rapidly concentrate a clinically relevant analyte of interest from a biological fluid. Our technology relies on the application of localized heat to a paper strip to evaporate off hundreds of microliters of liquid to concentrate the target analyte. This method can be used to enrich for a target analyte that is present at low concentrations within a biological fluid to enhance the sensitivity of downstream detection methods. We demonstrate our method by concentrating the tuberculosis-specific glycolipid, lipoarabinomannan (LAM), a promising urinary biomarker for the detection and diagnosis of tuberculosis. We show that the heat does not compromise the subsequent immunodetectability of LAM, and in 20 min, the tuberculosis biomarker was concentrated by nearly 20-fold in simulated urine. Our method requires only 500 mW of power, and sample flow is self-driven via capillary action. As such, our technology can be readily integrated into portable, battery-powered, instrument-free diagnostic devices intended for use in low-resource settings.

DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg- 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system.

The goal of the point-of-care (POC) sexually transmitted infection (STI) Diagnostics meeting was to review the state-of-the-art research and develop recommendations for the use of POC STI diagnostics. Experts from academia, government, nonprofit, and industry discussed POC diagnostics for STIs such as Chlamydia trachomatis, human papillomavirus, Neisseria gonorrhoeae, Trichomonas vaginalis, and Treponema pallidum. Key objectives included a review of current and emerging technologies, clinical and public health benefits, POC STI diagnostics in developing countries, regulatory considerations, and future areas of development. Key points of the meeting are as follows: (i) although some rapid point-of-care tests are affordable, sensitive, specific, easy to perform, and deliverable to those who need them for select sexually transmitted infections, implementation barriers exist at the device, patient, provider, and health system levels; (ii) further investment in research and development of point-of-care tests for sexually transmitted infections is needed, and new technologies can be used to improve diagnostic testing, test uptake, and treatment; (iii) efficient deployment of self-testing in supervised (ie, pharmacies, clinics, and so on) and/or unsupervised (ie, home, offices, and so on) settings could facilitate more screening and diagnosis that will reduce the burden of sexually transmitted infections; (iv) development of novel diagnostic technologies has outpaced the generation of guidance tools and documents issued by regulatory agencies; and (v) questions regarding quality management are emerging including the mechanism by which poor-performing diagnostics are removed from the market and quality assurance of self-testing is ensured.

Herein we show the development of a minimally instrumented paper-based molecular diagnostic for point of care detection of sexually transmitted infections caused by Chlamydia trachomatis. This new diagnostic platform incorporates cell lysis, isothermal nucleic acid amplification, and lateral flow visual detection using only pressure and heat sources, eliminating the need for expensive laboratory equipment. This paper-based test can be performed in less than one hour and has a clinically relevant limit of detection that is 100× more sensitive than current rapid immunoassays used for chlamydia diagnosis.

We report a disposable and highly effective polymeric microfluidic viral sample concentration device capable of increasing the concentration of virus in a human nasopharyngeal specimen more than one order of magnitude in less than 30 min without the use of a centrifuge. The device is fabricated using 3D maskless xurography method using commercially available polymeric materials, which require no cleanroom operations. The disposable components can be fabricated and assembled in five minutes. The device can concentrate a few milliliters (mL) of influenza virus in solution from tissue culture or clinical nasopharyngeal swab specimens, via reduction of the fluid volume, to tens of microliters (mL). The performance of the device was evaluated by nucleic acid extraction from the concentrated samples, followed by a real-time quantitative polymerase chain reaction (qRT-PCR). The viral RNA concentration in each sample was increased on average over 10-fold for both cultured and patient specimens compared to the starting samples, with recovery efficiencies above 60% for all input concentrations. Highly concentrated samples in small fluid volumes can increase the downstream process speed of on-chip nucleic acid extraction, and result in improvements in the sensitivity of many diagnostic platforms that interrogate small sample volumes.

Monitoring the adherence of patients taking highly-active anti-retroviral therapy (HAART) and Pre-Exposure Prophylaxis (PrEP) is a key step in treating an HIV infection. Since current monitor methods are non-direct there is a need for assays that directly verify the presence of active drug in the patient. In this work we modified an existing assay for the detection of cytosine to sense the presence of lamivudine (3TC) in spiked urine and emtricitabine (FTC) in water. 3TC and FTC is reacted with bromine and barium hydroxide to generate a colored precipitate in less than an hour indicating the presence of active drug. The current detection limit of the 3TC assay is 870uM in water and 2mM in urine. This assay can be applied in clinics to monitor patient adherence without the need for expensive equipment such as HPLC.

To advance the development of point-of-care technology (POCT), the National Institute of Biomedical Imaging and Bioengineering established the POCT Research Network (POCTRN), comprised of Centers that emphasize multidisciplinary partnerships and close facilitation to move technologies from an early stage of development into clinical testing and patient use. This paper describes the POCTRN and the three currently funded Centers as examples of academic-based organizations that support collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care., This paper describes the National Institute of Biomedical Imaging and Bioengineering's Point-of-Care Technologies Research Network, three Centers that, through multidisciplinary partnerships and close facilitation, are working to move point-of-care technologies from an early stage of development into clinical testing and patient use.

Traditional methods for identifying pathogens in bacteremic patients are slow (24–48+ h). This can lead to physicians making treatment decisions based on an incomplete diagnosis and potentially increasing the patient’s mortality risk. To decrease time to diagnosis, we have developed a novel technology that can recover viable bacteria directly from whole blood and identify them in less than 7 h. Our technology combines a sample preparation process with surface-enhanced Raman spectroscopy (SERS). The sample preparation process enriches viable microorganisms from 10 mL of whole blood into a 200 μL aliquot. After a short incubation period, SERS is used to identify the microorganisms. We further demonstrated that SERS can be used as a broad detection method, as it identified a model set of 17 clinical blood culture isolates and microbial reference strains with 100% identification agreement. By applying the integrated technology of sample preparation and SERS to spiked whole blood samples, we were able to correctly identify both Staphylococcus aureus and Escherichia coli 97% of the time with 97% specificity and 88% sensitivity.

Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications.

We present a new rapid prototyping method designed for simple fabrication of 3D microfluidics using a maskless direct writing technique on polymer substrates. The entire process is enabled by a commercial cutter plotter with [email protected] resolution precision and high speed. A CAD design of top and bottom microstructures is directly written on a polymer substrate using a cutter plotter after setting up the suitable force. The smallest channel width of [email protected] was obtained with the minimum force and [email protected] from the maximum. Also the written depth increased linearly with force from 30 to [email protected] Several 3D microfluidic devices are demonstrated using a maskless writing technique. The entire fabrication process from CAD layout to a final 3D device can be completed in 30min outside the clean room facilities.