At 3 a.m. Beijing time on July 21, the journal Science reported the heavy research results of Academician Charles M. Lieber (First Newsletter) on biological materials under the title "Ultraflexible endovascular probes for brain recording through micrometer-scale vasculature". Surprisingly, the first author and corresponding author of this paper turned out to be Zhang Anqi, the goddess of Fudan, who was popular all over the Internet.
From the initial idea, to the subsequent design, planning and main execution parts, mainly completed by her, Zhang Anqi is both the first author of this article and the co-corresponding author. Even all collaborators were found on their own. Years after the "stuffy" experiment, Zhang Anqi submitted her hard work to Science, and was accepted in just two months. 10 years ago, Zhang Anqi became popular on the Internet with the titles of "Mensa Girl" and "Fudan Student": she became the youngest TOEFL teacher at the age of 19 at the age of New Oriental, earning 50,000 yuan a month during the summer vacation and achieving "economic freedom"; Joined the "world's top IQ club" - Mensa Club, and served as the chief examiner of Mensa entrance test several times; taught himself programming in his sophomore year and became a nationally certified translator; At the age of 21, he held 5 SCI papers when he was an undergraduate... She is a typical "other people's child": Fudan University undergraduate + Harvard University master's degree + Stanford University postdoctoral all-round student.
In Zhang Anqi, it seems that you can see all the looks of a "perfect goddess": in 2010, Zhang Anqi was admitted to the Department of Materials Science of Fudan University with excellent results! In her freshman year, she took the GRE, TOEFL, Level 6 exams, and the results were close to full scores, and what was even more amazing was that as the only non-English major, she represented Shanghai in the Cambridge Business English Speech Competition in Asia, and finally won the third prize in the China final. At the age of 19, she became the youngest TOEFL teacher at New Oriental! Since her sophomore year, she has been a national certified translator and a student member of the China Translators Association! He later became a member of the Mensa Club and had five years of professional modeling experience. It is said that at that time, Zhang Anqi could earn 50,000 a month with her part-time job in English! However, not only English, but also Zhang Anqi is super good at scientific research! Fudan Materials Department has the first GPA, and has published 5 SCI papers as the first author, and has been invited to give oral reports at important academic conferences at home and abroad, such as the annual meeting of U21 International University Alliance, the National College Student Innovation and Entrepreneurship Annual Conference, and the Shanghai College Student Innovation Forum. He was also included in the "National Scholarship Winning Students' Style" as the only representative of Fudan University (a total of 72 students were selected by 50,000 national scholarship recipients).
Upon graduation, Anqi Zhang was admitted to Harvard University on a full scholarship! Studied under Charles M. Lieber, academician of the American Academy of Sciences!
Zhang Anqi and Charles Lieber In 2020, she entered the laboratory of Academician Bao Zhenan of Stanford University and began her postdoctoral work to continue the journey of scientific research dreams. As she says, "Research is a job that I decided to do for life, and after my PhD, I wanted to be a professor and concentrate on pursuing my academic ideals." ”
Talking about the impact of the popular Internet on him in the past, Zhang Anqi said that he has neither regrets nor nostalgia. "In those years, I received a lot of letters from my classmates, many of whom were girls. They told me that my story inspired them. This makes me very happy because although there are more girls doing scientific research now, there is still an imbalance between men and women. Just as Professor Bao Zhenan is my role model for women, I hope that I can encourage more female students to participate in scientific research. ”
Zhang Anqi and Bao Zhenan less than 3 weeks, the results were once again published in the Science sub-journal Gene-directed Chemical Synthesis Polymer! Can be specifically localized to the extracellular area of the surface membrane of living cells!
The highly complex structural and organizational properties of multicellular biological systems present challenges for researchers seeking to achieve cell-specific electrical interfaces without causing damage. The human brain contains about 86 billion neurons, connected by more than 100 trillion synapses that are essential for neuronal communication. Over the decades, there have been many attempts to shrink bioelectronics to the nanoscale and increase the scale of manufacturing processes, but until now, devices designed for the brain could only interact with hundreds of cells at a time and could not be cell-type-specific to tissues. Another way to solve this problem is to genetically program specific cells to build artificial structures with the desired morphology and function within a complete biological system. It has been proven that conductive polymers can be synthesized directly by electrochemical polymerization or on organisms and tissues with local oxidizing environments or oxidases. However, none of these methods has achieved the key goal of cell-specificity. The recently proposed method of genetically targeted chemical assembly (GTCA) is a first step in this direction. This method uses cell-specific genetic information to guide neurons to deposit polymeric materials with various conductivity properties in situ. Through electrophysiological and behavioral analysis, it was confirmed that gene-directed assembled functional polymers reshaped membrane properties without the need to use transgenic animal lines and successfully modulated cell type-specific behavior in freely active animals.
Despite the initial success of this approach, there was an important limitation previously used in proof-of-concept systems: the reaction center was not specifically localized to the outside of the cell membrane. This feature limits further applications of biomanufacturing platforms. This is because living cells with intact membranes are generally not permeable to macromolecular precursors or materials; Thus, insufficient display of enzymes on the membrane may lead to low yields of chemical assembly in living systems. Second, increasing the number of enzymes that catalyze the reaction (utilizing extracellular space) is expected to reduce the concentration of reagents required to set the reaction conditions, thereby improving biocompatibility. Third, positioning the reaction in extracellular space may attenuate the adverse effects on the process of intracellular chemical reactions; Previous reports have confirmed that certain intracellular polymerization reactions can be toxic to cells and can induce apoptosis. Therefore, developing a method that can efficiently place the reaction center completely in the extracellular space (the outside of the membrane) remains a challenge. On August 9, 2023, Karl Deisseroth, Academician of Stanford University and father of optogenetics, collaborated to develop a next-generation gene-directed chemical assembly (GTCA) method that highly localized the expression of enzymes targeting the plasma membrane of primary neurons while achieving minimal intracellular retention. Polymers synthesized outside the cell in this way form dense clusters around the membranes of designated living neurons, and neurons survive the polymerization reaction. This membrane localization method proved to be easily adapted to anchor other proteins, making it possible to explore diverse alternative GTCA strategies. The work was published in the latest issue of Science Advances in a paper titled "Genetically targeted chemical assembly of polymers specifically localized extracellularly to surface membranes of living neurons." The first author is Dr. Angel Zhang.
In situ GTCA Figure 1A of neuronal surface polymers shows the construction design for expressing the membrane-shown form of HRP in primary neurons, where targeted neurons are expected to express not only HRP shown by the outer membrane but also the cytoplasmic yellow fluorescent protein YFP, and the location of HRP can be determined by staining with antibodies targeting the FLAG tag. After the addition of small molecule polymer precursors and H2O2, the HRPs shown by the membrane act as reaction centers, promoting oxidative radical polymerization on targeted neurons. Due to the low solubility of the resulting polymers, these synthetic polymers are expected to be localized not only outside the cell, but also deposited on the targeted cell membrane. Here, the authors selected two polymer precursors, N-phenyl-p-phenylenediamine (aniline dimer) and 3,3'-diaminobiphenyl (DAB monomer), for depositing conductive polymer polyaniline (PANI) and non-conductive polymer poly(3,3'-diaminobiphenyl) (PDAB), respectively (Figure 1B).
Figure 1. Schematic diagram of the next generation of GTCA in neurons. To quantitatively compare the resulting membrane expression profiles, the authors performed antibody staining on non-detergent-permeated or detergent-permeated cells (Figure 2A). In non-detergent-permeable cells, membrane-anchored enzymes can be selectively detected, while in detergent-permeated cells, enzymes can be strongly labeled intracellular and extracellular.
Figure 2. Membrane localization of peroxidase and evaluation of peroxidase activity. In situ polymer deposition and morphological characterization, the authors then performed a polymerization of PANI and PDAB on HRP(+) and HRP(-) neurons. Brightfield images show that HRP(+) neurons exhibit dark reaction products, while HRP(-) neurons do not (Figure 3A). The reaction progression is quantified as a decrease in the brightness of the cells relative to the background (Figure 3B). PANI forms densely distributed aggregates, while PDAB forms a thin, uniform coating with uneven aggregates scattered on it. The selectivity of the response can be easily assessed between transfected neurons (i.e., cells expressing strong cytoplasmic YFP signaling) and non-transfected neurons in the same field of view (i.e., cells with low or no YFP signal). When PANI and PDAB polymerization is performed on active HRP-CD2(+) [i.e., HRP(+)] neurons, transfected cells become much darker than neighboring non-transfected cells (Figure 3C). After performing the same response on HRP-CD8(+) cells (Figure 3C), a statistical comparison of the difference in brightness ratio (Figure 3D) showed that HRP-CD2(+) neurons had significantly better selectivity compared to HRP-CD8(+) neurons.
Figure 3. In situ genes on living neuronal membranes target polymer deposition. The authors confirmed the morphology of two polymer deposits by scanning electron microscopy (SEM) imaging (Figure 4A). HRP(+)/PANI neurons form dense clusters around the neurosomes and neurites. In contrast, HRP(+) cells not exposed to PANI formed a smooth membrane surface. The localization specificity of this gene targeting method was confirmed by further comparison with SEM images of HRP(-) cells. The authors also used atomic force microscopy (AFM) to characterize the height of the PANI particles on the neuromembrane and the Delyaguin-Müller-Topolov modulus distribution (Figure 4B). The modulus of the membrane region between the particles is much higher than that of the unmodified film, which indicates that a thin layer of PANI is uniformly coated between the particles.
Figure 4. Morphological and mechanical properties of in situ deposited polymers. Cell viability assays and exploration of alternative GTCA strategies The authors used cell viability assays to monitor HRP(+) cells before and after polymerization (Figure 5D), evaluated the YFP signal of active HRP(+) cells, and then stained the cells with ethyldimer (EthD-1) after PANI and PDAB deposition. The location of HRP(+) cells is marked with white dots in the EthD-1 channel (Figure 5D); The absence of overlap with red blood cells indicates that HRP(+) neurons are still alive after polymerization.
Figure 5. Spectral characterization and cell viability assays of in situ deposited polymers. In addition to peroxidase-controlled oxidative polymerization, the authors explore alternative strategies to advance gene-targeted chemical synthesis (GTCA), all benefiting from this powerful outer membrane anchoring CD2 strategy. The authors tested and successfully demonstrated two conjugated GTCA strategies for cell-specific synthesis: a gene-targeted light-controlled oxidative polymerization strategy (Figure 6, A-D) and a gene-targeted conjugate of presynthetic material (Figure 6, E-G). The authors experimentally confirmed that both strategies enable selective anchorage of the material to cell membranes.
Figure 6. Explore alternative GTCA strategies. This work addresses a key limitation of the GTCA platform by designing and implementing a method for synthesizing functional materials for genetically specified cells that allows key enzymes to be precisely and smoothly targeted into the extracellular space while the enzyme (and reaction products) remain confined to the plasma membrane of interest. The study found that polymers synthesized in situ on primary neurons selectively formed dense clusters outside the cells of the surface plasma membrane, and that neurons remained alive after completing the synthesis reaction in this way. This method locates the reaction in extracellular space, which in addition to supporting biocompatibility, increases reaction yields and reduces reactant concentrations. --Cellulose Recommended ---- Recommendation Number--
https://www.science.org/doi/full/10.1126/sciadv.adi1870 Source: Frontiers in Polymer Science