Multiplexing Capability Research Articles

Development of an integrated sample amplification control for salivary point-of-care pathogen testing

BackgroundThe COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed. ResultsWe developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/μL of saliva within 30 min without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples. SignificanceIACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

Surface-Enhanced Raman Scattering-Based Surface Chemotaxonomy: Combining Bacteria Extracellular Matrices and Machine Learning for Rapid and Universal Species Identification.

Rapid, universal, and accurate identification of bacteria in their natural states is necessary for on-site environmental monitoring and fundamental microbial research. Surface-enhanced Raman scattering (SERS) spectroscopy emerges as an attractive tool due to its molecule-specific spectral fingerprinting and multiplexing capabilities, as well as portability and speed of readout. Here, we develop a SERS-based surface chemotaxonomy that uses bacterial extracellular matrices (ECMs) as proxy biosignatures to hierarchically classify bacteria based on their shared surface biochemical characteristics to eventually identify six distinct bacterial species at >98% classification accuracy. Corroborating with in silico simulations, we establish a three-way inter-relation between the bacteria identity, their ECM surface characteristics, and their SERS spectral fingerprints. The SERS spectra effectively capture multitiered surface biochemical insights including ensemble surface characteristics, e.g., charge and biochemical profiles, and molecular-level information, e.g., types and numbers of functional groups. Our surface chemotaxonomy thus offers an orthogonal taxonomic definition to traditional classification methods and is achieved without gene amplification, biochemical testing, or specific biomarker recognition, which holds great promise for point-of-need applications and microbial research.

Plasmonic Nanoparticle-Enhanced Optical Techniques for Cancer Biomarker Sensing.

This review summarizes recent advances in leveraging localized surface plasmon resonance (LSPR) nanotechnology for sensitive cancer biomarker detection. LSPR arising from noble metal nanoparticles under light excitation enables the enhancement of various optical techniques, including surface-enhanced Raman spectroscopy (SERS), dark-field microscopy (DFM), photothermal imaging, and photoacoustic imaging. Nanoparticle engineering strategies are discussed to optimize LSPR for maximum signal amplification. SERS utilizes electromagnetic enhancement from plasmonic nanostructures to boost inherently weak Raman signals, enabling single-molecule sensitivity for detecting proteins, nucleic acids, and exosomes. DFM visualizes LSPR nanoparticles based on scattered light color, allowing for the ultrasensitive detection of cancer cells, microRNAs, and proteins. Photothermal imaging employs LSPR nanoparticles as contrast agents that convert light to heat, producing thermal images that highlight cancerous tissues. Photoacoustic imaging detects ultrasonic waves generated by LSPR nanoparticle photothermal expansion for deep-tissue imaging. The multiplexing capabilities of LSPR techniques and integration with microfluidics and point-of-care devices are reviewed. Remaining challenges, such as toxicity, standardization, and clinical sample analysis, are examined. Overall, LSPR nanotechnology shows tremendous potential for advancing cancer screening, diagnosis, and treatment monitoring through the integration of nanoparticle engineering, optical techniques, and microscale device platforms.

Quantum optical memory for entanglement distribution

Optical photons are powerful carriers of quantum information, which can be delivered in free space by satellites or in fibers on the ground over long distances. Entanglement of quantum states over long distances can empower quantum computing, quantum communications, and quantum sensing. Quantum optical memories are devices designed to store quantum information in the form of stationary excitations, such as atomic coherence, and are capable of coherently mapping these excitations to flying qubits. Quantum memories can effectively store and manipulate quantum states, making them indispensable elements in future long-distance quantum networks. Over the past two decades, quantum optical memories with high fidelities, high efficiencies, long storage times, and promising multiplexing capabilities have been developed, especially at the single-photon level. In this review, we introduce the working principles of commonly used quantum memory protocols and summarize the recent advances in quantum memory demonstrations. We also offer a vision for future quantum optical memory devices that may enable entanglement distribution over long distances.

Ultrasensitive, Multiplexed Buoyant Sensor for Monitoring Cytokines in Biofluids.

Multiplexed quantification of low-abundance protein biomarkers in complex biofluids is important for biomedical research and clinical diagnostics. However, in situ sampling without perturbing biological systems remains challenging. In this work, we report a buoyant biosensor that enables in situ monitoring of protein analytes at attomolar concentrations with a 15 min temporal resolution. The buoyant biosensor implemented with fluorescent nanolabels enabled the ultrasensitive and multiplexed detection and quantification of cytokines. Implementing the biosensor in a digital manner (i.e., counting the individual nanolabels) further improves the low detection limit. We demonstrate that the biosensor enables the detection and quantification of the time-varying concentrations of cytokines (e.g., IL-6 and TNF-α) in macrophage culture media without perturbing the live cells. The easy-to-apply biosensor with attomolar sensitivity and multiplexing capability can enable an in situ analysis of protein biomarkers in various biofluids and tissues to aid in understanding biological processes and diagnosing and treating diverse diseases.

Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein.

Thanks to its ease, speed, and sensitivity, CRISPR-based nucleic acid detection has been increasingly explored for molecular diagnostics. However, one of its major limitations is lack of multiplexing capability because the detection relies on the trans-cleavage activity of the Cas protein, which necessitates the use of multiple orthogonal Cas proteins for multiplex detection. Here we report the development of a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein (Cas12a). This method, termed as CRISPR-TMSD, integrates the toehold-mediated strand displacement (TMSD) reaction, and the cis-cleavage activity of the Cas protein and multiplexed detection are achieved using a single Cas protein owing to the use of target-specific reporters. A set of computational simulation toolkits was used to design the TMSD reporter, allowing for highly sensitive and specific identification of target sequences. In combination with the recombinase polymerase amplification (RPA), the detection limit can reach as low as 1 copy/μL. As proof of concept, CRISPR-TMSD was subsequently used to detect an oncogenic gene and SARS-CoV-2 RNA with a single-nucleotide resolution. This work represents a conceptually new strategy for designing a CRISPR-based diagnostic system and has great potential to expand the application of CRISPR-based diagnostics.

RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses.

The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

Dual-Core Ce: YAG-Derived Silicate Scintillation Fiber for Real-Time X-Ray Detection

A dual-core Ce:YAG-derived scintillation fiber (DCSF) was proposed conceptually, simulated numerically, and prepared experimentally by the modified molten core method (MCM) for the first time, of which, the core material is amorphous yttrium aluminosilicate glasses and the cladding material SiO2. Theoretically, it is found that such kind of DCSF exhibits very low inter-core crosstalk with the help of finite element simulation analysis, which may result from the high index difference of ∼0.097 between core and cladding. The average decay lifetime of the prepared DCSF are 31.4 ns and 36 ns at 350 nm and 500 nm, which are shorter than those of its Ce:YAG bulk crystal counterpart. Radiation monitoring was demonstrated by an all-fiber detection system. The radiation response of the DCSF bundle was also investigated, whose radio-luminescence response intensity is ∼1.66 and ∼1.12 times higher than that of the single-core fiber bundles with similar core-diameter and with similar core-area, respectively. In conclusion, with radiation detection performance improvement enabled by space division multiplexing capability, the DCSF not only provides a solution for the dilemma between low transmission loss and high optical yield, but also makes a substantial step towards the development of practical and reliable radiation detectors.

Short tandem repeat profiling via next-generation sequencing for cell line authentication.

Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple passages (often indefinitely), allowing multiple experiments to be performed. However, over time, cell line identity and purity can be compromised by human errors. Cross-contamination from other cell lines and complete misidentification are both possible. Routine cell line authentication is a necessary preventive measure and has become a requirement for many funding applications and publications. Short tandem repeat (STR) profiling is the most common method for cell line authentication and is usually carried out using standard polymerase chain reaction-capillary electrophoresis analysis (STR-CE). Here, we evaluated next-generation sequencing (NGS)-based STR profiling of human and mouse cell lines at 18 and 15 loci, respectively, in a high-throughput format. Using the Python program STRight, we demonstrate that NGS-based analysis (STR-NGS) is superior to standard STR-CE in terms of the ability to report the sequence context of repeat motifs, sensitivity and flexible multiplexing capability. STR-NGS is thus a valuable alternative for cell line authentication.

Stretching boundaries in neurophysiological monitoring

AbstractThe most prevalent among nervous system tumors significantly jeopardize patient health. For nerve integrity preservation after tumor removal, continuous intraoperative neurophysiological monitoring (CINM) is indispensable during microsurgery. The paper highlights the articles about the development of a system that employs soft and stretchable organic electronic materials for CINM. This innovative system harnesses soft and stretchable organic electronic materials and deploys conductive polymer electrodes with low impedance and modulus. These electrodes facilitate uninterrupted near‐field action potential recording during surgery, resulting in enhanced signal‐to‐noise ratios and reduced invasiveness. Additionally, the system's multiplexing capabilities enable precise nerve localization, even in the absence of anatomical landmarks.

UAV-Assisted Wideband Terahertz Wireless Communications with Time-Delay Phased UPA under Beam Squint

Future Unmanned Aerial Vehicle (UAV)-assisted wireless communication systems are expected to utilize wide bandwidths available at terahertz (THz) frequencies to enhance system throughput. To compensate for the severe path loss in the THz band, it is essential to have a multitude of antennas in the UAV to generate narrow beams for directional transmission. However, narrow beams severely limit its spatial coverage, which greatly affects the efficiency of large-scale access UAV-assisted THz systems. Moreover, the combination of massive antennas and large bandwidth at THz makes the misalignment of the beams caused by beam squint non-negligible and also high energy consumption. UAV-assisted communication technology can effectively increase spatial coverage and provide reliable LoS communication links. In addition, reducing the number of radio frequency (RF) chains while ensuring the number of transmitted data streams and space division multiplexing capability is also an effective way to reduce energy consumption in the UAV communication. In this paper, a single RF chain uniform planar array (UPA) with true-time-delays (TTDs) is equipped on the UAV to achieve two dimensional (2D) beams and split spatial beams to improve transmission efficiency. We analyze the 2D beam squint of the UPA and design a time-delay phased UPA for UAV-assisted THz communication systems. By introducing TTDs between the single RF chain and phase shifters, the beam squint can be controlled flexibly by introducing the delay between each antenna. When TTDs are arranged in both the horizontal and vertical dimensions, the coverage of the beams becomes more complicated compared to uniform linear arrays (ULA). Simulation results show that the proposed time-delay phased UPA can achieve better performance in both single-beam and multi-beam modes for single user and multi-user scenarios compared with conventional phased UPA, respectively. In addition, we propose frequency division beam multiple access (FDBMA) multi access technology, which achieves more efficient multi access by reusing resources from different frequency beam pairs. Finally, the results also show that the enlargement of the beamwidth through the proposed FDBMA strategy can also increase the performance in multi-user scenarios.

Fibre Bragg Grating Sensors for Condition Monitoring of High-Voltage Assets: A Review

The high-voltage (HV) assets in the existing power transmission network will experience increased electrical, thermal, environmental and mechanical stresses and, therefore, robust condition monitoring is critical for power system reliability planning. Fibre Bragg grating (FBG) sensors offer a promising technology in HV applications due to their immunity to electromagnetic interference and multiplexing capability. This paper reviews the current technology readiness levels of FBG sensors for condition monitoring of transformers, transmission lines, towers, overhead insulators and power cables, with the aim of stimulating further development and deployment of fibre-based HV asset management systems. Currently, there are several reported cases of FBG sensors used for condition monitoring of HV assets in the field, proving their feasibility for long-term use in the power grid. The review shows that FBG technology is versatile and can facilitate multi-parameter measurements, which will standardise the demodulation equipment and reduce challenges with integrating different sensing technologies.

Multiplex genotyping of SNPs in genomic DNA via hydrogel-based assay mediated with MutS and polyethylene glycol

The simultaneous genotyping of multiple single nucleotide polymorphisms (SNPs) in genomic DNA derived from organisms holds significant potential for applications such as precision medicine and food product authentication. However, conventional assay technologies including qPCR-based techniques, microarrays, and hydrogel-based assays face limitations in efficient multiplexing of SNPs, particularly for large-size DNA beyond kilobase scales, due to constraints in multiplex capability, specificity, or sensitivity. In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed ∼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.

Integrated Flexible Microscale Mechanical Sensors Based on Cascaded Free Spectral Range-Free Cavities.

Photonic mechanical sensors offer several advantages over their electronic counterparts, including immunity to electromagnetic interference, increased sensitivity, and measurement accuracy. Exploring flexible mechanical sensors on deformable substrates provides new opportunities for strain-optical coupling operations. Nevertheless, existing flexible photonics strategies often require cumbersome signal collection and analysis with bulky setups, limiting their portability and affordability. To address these challenges, we propose a waveguide-integrated flexible mechanical sensor based on cascaded photonic crystal microcavities with inherent deformation and biaxial tensile state analysis. Leveraging the advanced multiplexing capability of the sensor, for the first time, we successfully demonstrate 2D shape reconstruction and quasi-distributed strain sensing with 110 μm spatial resolution. Our microscale mechanical sensor also exhibits exceptional sensitivity with a detected force level as low as 13.6 μN in real-time measurements. This sensing platform has potential applications in various fields, including biomedical sensing, surgical catheters, aircraft and spacecraft engineering, and robotic photonic skin development.

Two Decades of Arrayed Imaging Reflectometry for Sensitive, High-Throughput Biosensing.

Arrayed imaging reflectometry (AIR), first introduced in 2004, is a thin-film interference sensor technique that optimizes optical properties (angle of incidence, polarization, substrate refractive index, and thickness) to create a condition of total destructive interference at the surface of a silicon substrate. The advantages of AIR are its sensitivity, dynamic range, multiplex capability, and high-throughput compatibility. AIR has been used for the detection of antibodies against coronaviruses, influenza viruses, Staphylococcus aureus, and human autoantigens. It has also shown utility in detection of cytokines, with sensitivity comparable to bead-based and ELISA assays. Not limited to antibodies or antigens, mixed aptamer and protein arrays as well as glycan arrays have been employed in AIR for differentiating influenza strains. Mixed arrays using direct and competitive inhibition assays have enabled simultaneous measurement of cytokines and small molecules. Finally, AIR has also been used to measure affinity constants, kinetic and at equilibrium. In this review, we give an overview of AIR biosensing technologies and present the latest AIR advances.

Stellar metallicity from optical and UV spectral indices: Test case for WEAVE-StePS

Context. The upcoming generation of optical spectrographs on four meter-class telescopes, with their huge multiplexing capabilities, excellent spectral resolution, and unprecedented wavelength coverage, will provide high-quality spectra for thousands of galaxies. These data will allow us to examine of the stellar population properties at intermediate redshift, an epoch that remains unexplored by large and deep surveys. Aims. We assess our capability to retrieve the mean stellar metallicity in galaxies at different redshifts and signal-to-noise ratios (S/N), while simultaneously exploiting the ultraviolet (UV) and optical rest-frame wavelength coverage. Methods. The work is based on a comprehensive library of spectral templates of stellar populations, covering a wide range of age and metallicity values and built assuming various star formation histories, to cover an observable parameter space with diverse chemical enrichment histories and dust attenuation. We took into account possible observational errors, simulating realistic observations of a large sample of galaxies carried out with WEAVE at the William Herschel Telescope at different redshifts and S/N values. We measured all the available and reliable indices on the simulated spectra and on the comparison library. We then adopted a Bayesian approach to compare the two sets of measurements in order to obtain the probability distribution of stellar metallicity with an accurate estimate of the uncertainties. Results. The analysis of the spectral indices has shown how some mid-UV indices, such as BL3580 and Fe3619, can provide reliable constraints on stellar metallicity, along with optical indicators. The analysis of the mock observations has shown that even at S/N = 10, the metallicity can be derived within 0.3 dex, in particular, for stellar populations older than 2 Gyr. The S/N value plays a crucial role in the uncertainty of the estimated metallicity and so, the differences between S/N = 10 and S/N = 30 are quite large, with uncertainties of ~0.15 dex in the latter case. On the contrary, moving from S/N = 30 to S/N = 50, the improvement on the uncertainty of the metallicity measurements is almost negligible. Our results are in good agreement with other theoretical and observational works in the literature and show how the UV indicators, coupled with classic optical ones, can be advantageous in constraining metallicities. Conclusions. We demonstrate that a good accuracy can be reached on the spectroscopic measurements of the stellar metallicity of galaxies at intermediate redshift, even at low S/N, when a large number of indices can be employed, including some UV indices. This is very promising for the upcoming surveys carried out with new, highly multiplexed, large-field spectrographs, such as StePS at the WEAVE and 4MOST, which will provide spectra of thousands of galaxies covering large spectral ranges (between 3600 and 9000 Å in the observed frame) at relatively high S/N (>10 Å−1).

In situ synthesis of gold nanoparticles on layered double hydroxide nanoparticles for multiplexing molecular imaging of single cells

Quasi-spherical gold nanoparticles (AuNPs) are the most widely used surface-enhanced Raman scattering (SERS) nanotags. However, their surface plasmon resonance properties are significantly restricted by their poor colloidal stability in biological media. Herein, we developed a new strategy for in situ synthesis of AuNPs on layered double hydroxide (LDH) nanoparticles to improve the colloidal stability of AuNPs for SERS imaging with multiplexing capability using single laser excitation. The hybridized LDH-AuNPs were constructed via in situ growth of AuNPs on lactic acid-LDH nanoparticles. The size of AuNPs was tuned by changing the concentration of LDH. The LDH-AuNPs were further functionalized with Raman reporter molecules (Ra) to obtain LDH-AuNPs-Ra as SERS nanotags, which were examined in terms of the colloidal stability and photostability in biological media as well as cellular uptake in J774A.1 macrophage cell culture by single cell mapping via confocal Raman microscopy. The cellular uptake of multiple LDH-AuNPs-Ra functionalized with different Raman reporter molecules were further investigated, where the multiplexing capability of SERS assay endows simultaneous detection of multiple LDH-AuNPs-Ra distribution after cellular uptake for cell imaging. In summary, the LDH-supported in situ synthesis of AuNPs have shown the merits of stabilizing AuNPs by using LDH for SERS imaging, as well as demonstrating the multiplexing molecular imaging of single cells. This work thus provides a promising strategy and a new bioimaging nanoplatform for various biomedical studies in the near future.

A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing.

Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL- 1 level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1h, making it suitable for low-resource settings.

Barcoded multiple displacement amplification for high coverage sequencing in spatial genomics

Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.